Heterocyclic thioamide derivatives

ABSTRACT

It has been discovered that compounds of the formula:  
                 
 
     and the pharmaceutically acceptable salts and esters thereof wherein X and Y are as defined below, inhibit the binding of VCAM-1 to VLA-4 and are useful in treating inflammation associated with chronic inflammatory diseases such as rheumatoid arthritis (RA), multiple sclerosis, (MS), asthma, and inflammatory bowel disease (IBD).

BACKGROUND OF THE INVENTION

[0001] Vascular cell adhesion molecule-1 (VCAM-1), a member of theimmunoglobulin (Ig) supergene family, is expressed on activated, but notresting, endothelium. The integrin VLA-4 (a₄b₁), which is expressed onmany cell types including circulating lymphocytes, eosinophils,basophils, and monocytes, but not neutrophils, is the principal receptorfor VCAM-1. Antibodies to VCAM-1 or VLA-4 can block the adhesion ofthese mononuclear leukocytes, as well as melanoma cells, to activatedendothelium in vitro. Antibodies to either protein have been effectiveat inhibiting leukocyte infiltration and preventing tissue damage inseveral animal models of inflammation. Anti-VLA-4 monoclonal antibodieshave been shown to block T-cell emigration in adjuvant-inducedarthritis, prevent eosinophil accumulation and bronchoconstriction inmodels of asthma, and reduce paralysis and inhibit monocyte andlymphocyte infiltration in experimental autoimmune encephalitis (EAE).Anti-VCAM-1 monoclonal antibodies have been shown to prolong thesurvival time of cardiac allografts. Recent studies have demonstratedthat anti-VLA-4 mAbs can prevent insulitis and diabetes in non-obesediabetic mice, and significantly attenuate inflammation in thecotton-top tamarin model of colitis.

[0002] Thus, compounds which inhibit the interaction betweenα4-containing integrins and VCAM-1 will be useful as therapeutic agentsfor the treatment of inflammation resulting from chronic inflammatorydiseases such as rheumatoid arthritis, multiple sclerosis (MS), asthma,and inflammatory bowel disease (IBD).

CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application is related to:

[0004] Chen, et al. U.S. Ser. No. 09/138,353 filed Aug. 21, 1998, Title:N-ALKANOYLPHENYLALANINE DERIVATIVES; and

[0005] Chen, et al. U.S. Ser. No. 09/137,798 filed Aug. 21, 1998, Title:N-AROYLPHENYLALANINE DERIVATIVES.

SUMMARY OF THE INVENTION

[0006] It has been discovered that compounds of the formula:

[0007] and the pharmaceutically acceptable salts and esters thereofwherein X and Y are as defined below, inhibit the binding of VCAM-1 toVLA-4 and are useful in treating inflammation associated withchronicinflammatory diseases such as rheumatoid arthritis (RA), multiplesclerosis, (MS), asthma, and inflammatory bowel disease (IBD).

DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention is directed to the discovery that compoundsof the formula:

[0009] and the pharmaceutically acceptable salts and esters thereof,inhibit the binding of VCAM-1 to VLA-4 and are useful in treatinginflammation associated with chronic inflammatory diseases such asrheumatoid arthritis (RA), multiple sclerosis, (MS), asthma, aninflammatory bowel disease (IBD).

[0010] In accordance with the invention, X is a group X-1, X-2 or X-3 asdescribed below. Y is a group Y-1, Y-2 or Y-3 as described below.

[0011] The group X-1 is of the formula:

[0012] wherein:

[0013] R₁₅ is halogen, nitro, lower alkyl sulfonyl, cyano, lower alkyl,lower alkoxy, lower alkoxycarbonyl, carboxy, lower alkyl aminosulfonyl,perfluorolower alkyl, lower alkylthio, hydroxy lower alkyl, alkoxy loweralkyl, lower alkylthio lower alkyl, lower alkylsulfinyl lower alkyl,lower alkylsulfonyl lower alkyl, lower alkylsulfinyl, lower alkanoyl,aroyl, aryl, aryloxy;

[0014] R₁₆ is hydrogen, halogen, nitro, cyano, lower alkyl, OH,perfluorolower alkyl, or lower alkylthio.

[0015] The groups R₁₅ and R₁₆ are preferably independently hydrogen,lower alkyl, nitro, halogen (especially chloro or fluoro),perfluoromethyl, or cyano for R₁₆, and lower alkyl, nitro, halogen(especially chloro or fluoro), perfluoromethyl, or cyano for R₁₅.

[0016] It is preferred that groups selected as R₁₅, or R₁₅ and R₁₆, beelectron-deficient as defined below.

[0017] X-2 is a group of the formula:

[0018] wherein Het is a 5- or 6-membered heteroaromatic ring containing1, 2 or 3 heteroatoms selected from N,O, and S,

[0019] or

[0020] Het is a 9- or 10-membered bicyclic heteroaromatic ringcontaining 1, 2, 3 or 4 heteroatoms selected from O, S, and N;

[0021] R₁₅ and R₁₆ are as above, and

[0022] R₃₀ is hydrogen or lower alkyl; and p is an integer from 0 to 1.

[0023] Het is preferably a 5- or 6-membered monocyclic heteroaromaticring containing 1, 2 or 3 nitrogens, or a nitrogen and a sulfur, or anitrogen and an oxygen. When Het is a bicyclic heteroaromatic ring, itpreferably contains from 1 to 3 nitrogens as the heteroatoms. R₁₅ ispreferably, nitro, lower alkyl sulfonyl, cyano, lower alkyl, loweralkoxy, perfluorolower alkyl, lower alkylthio, lower alkanoyl, or aryl(especially unsubstituted phenyl); R₁₆ is preferably hydrogen, halogen,nitro, cyano, lower alkyl, perfluoro lower alkyl; and R₃₀, when present,is preferably hydrogen or lower alkyl.

[0024] The group X-3 is of the formula:

[0025] wherein:

[0026] R₁₈ is aryl, heteroaryl,

[0027] R₁₉ is substituted or unsubstituted lower alkyl, aryl,heteroaryl, arylalkyl, heteroaryl alkyl, and

[0028] R₂₀ is substituted or unsubstituted lower alkanoyl or aroyl

[0029] R₁₈ is preferably phenyl. R₁₉ is preferably lower alkyl, which isunsubstituted or substituted by pyridyl or phenyl. R₂₀ is preferablylower alkanoyl

[0030] Y is a group of formula Y-1, Y-2, or Y-3 wherein:

[0031] Y-1 is a group of the formula:

[0032] wherein:

[0033] R₂₂ and R₂₃ are independently hydrogen, lower alkyl, loweralkoxy, cycloalkyl, aryl, arylalkyl, nitro, cyano, lower alkylthio,lower alkylsulfinyl, lower alkyl sulfonyl, lower alkanoyl, halogen, orperfluorolower alkyl and at least one of R₂₂ and R₂₃ is other thanhydrogen, and

[0034] R₂₄ is hydrogen, lower alkyl, lower alkoxy, aryl, nitro, cyano,lower alkyl sulfonyl, or halogen

[0035] Y-2 is a group of the formula

[0036] Het is a five or six membered heteroaromatic ring bonded via acarbon atom wherein said ring contains one, two or three heteroatomsselected from the group consisting of N, O and S and R₃₀ and R₃₁ areindependently hydrogen, lower alkyl, cycloalkyl, halogen, cyano,perfluoroalkyl, or aryl and at least one of R₃₀ and R₃₁ is adjacent tothe point of attachment, p is an integer of from 0 to 1.

[0037] Y-3 is a 3-7 membered ring of the formula:

[0038] wherein:

[0039] R₂₅ is lower alkyl, unsubstituted or fluorine substituted loweralkenyl, or a group of formula R₂₆—(CH₂)₃—, R₂₆ is aryl, heteroaryl,azido, cyano, hydroxy, lower alkoxy, lower alkoxycarbonyl, loweralkanoyl, lower alkylthio , lower alkyl sulfonyl, lower alkyl sulfinyl,perfluoro lower alkanoyl, nitro, or R₂₆ is a group of formula —NR₂₈R₂₉,

[0040] wherein

[0041] R₂₈ is H or lower alkyl,

[0042] R₂₉ is hydrogen, lower alkyl, lower alkoxycarbonyl, loweralkanoyl, aroyl, perfluoro lower alkanoylamino, lower alkyl sulfonyl,lower alkylaminocarbonyl, arylaminocarbonyl,

[0043] or

[0044] R₂₈ and R₂₉ taken together form a 4, 5 or 6-membered saturatedcarbocyclic ring optionally containing one heteroatom selected from O,S, and N with the carbon atoms in the ring being unsubstituted orsubstituted by lower alkyl or halogen,

[0045] Q is —(CH₂)_(f)O—, —(CH₂)_(f)S—, —(CH₂)_(f)N(R₂₇)—, or—(CH₂)_(f)—,

[0046] R₂₇ is H, lower alkyl, aryl, lower alkanoyl, aroyl or loweralkoxycarbonyl,

[0047] e is an integer from 0 to 4, and f is an integer from 0 to 3; thedotted bond is optionally hydrogenated.

[0048] This invention is directed to a compound of the formula:

[0049] wherein X is a group of the formula

[0050] wherein:

[0051] R₁₅ is halogen, nitro, lower alkyl sulfonyl, cyano, lower alkyl,lower alkoxy, lower alkoxycarbonyl, carboxy, lower alkyl aminosulfonyl,perfluorolower alkyl, lower alkylthio, hydroxy lower alkyl, alkoxy loweralkyl, lower alkylthio lower alkyl, lower alkylsulfinyl lower alkyl,lower alkylsulfonyl lower alkyl, lower alkylsulfinyl, lower alkanoyl,aroyl, aryl, aryloxy;

[0052] R₁₆ is hydrogen, halogen, nitro, cyano, lower alkyl, OH,perfluorolower alkyl, or lower alkylthio; or

[0053] X is a group of formula X-2

[0054] wherein Het is a 5- or 6-membered heteroaromatic ring containing1, 2 or 3 heteroatoms selected from N,O, and S, or

[0055] Het is a 9- or 10-membered bicyclic heteroaromatic ringcontaining 1, 2, 3 or 4 heteroatoms selected from O, S, and N;

[0056] R₁₅ and R₁₆ are as above;

[0057] R₃₀ is hydrogen or lower alkyl; and p is an integer from 0 to 1or X is a group of formula X-3

[0058] wherein:

[0059] R₁₈ is aryl, heteroaryl, aryl lower alkyl, heteroaryl lower alkyl

[0060] R₁₉ is substituted or unsubstituted lower alkyl, aryl,heteroaryl, arylalkyl, heteroaryl alkyl, and

[0061] R₂₀ is substituted or unsubstituted lower alkanoyl or aroyl;

[0062] and Y is a group of formula Y-1

[0063] wherein:

[0064] R₂₂ and R₂₃ are independently hydrogen, lower alkyl, loweralkoxy, cycloalkyl, aryl, arylalkyl, nitro, cyano, lower alkylthio,lower alkylsulfinyl, lower alkyl sulfonyl, lower alkanoyl, halogen, orperfluorolower alkyl and at least one of R₂₂ and R₂₃ is other thanhydrogen, and

[0065] R₂₄ is hydrogen, lower alkyl, lower alkoxy, aryl, nitro, cyano,lower alkyl sulfonyl, or halogen;

[0066] or Y-2 is a group of the formula:

[0067] Het is a five or six membered heteroaromatic ring bonded via acarbon atom wherein said ring contains one, two or three heteroatomsselected from the group consisting of N, O and S and R₃₀ and R₃₁ areindependently hydrogen, lower alkyl, cycloalkyl, halogen, cyano,perfluoroalkyl, or aryl and at least one of R₃₀ and R₃₁ is adjacent tothe point of attachment; p is an integer of from 0 to 1;

[0068] or Y is a group of formula Y-3

[0069] wherein:

[0070] R₂₅ is lower alkyl, unsubstituted or fluorine substituted loweralkenyl, or a group of formula R₂₆—(CH₂)_(e)—, R₂₆ is aryl, heteroaryl,azido, cyano, hydroxy, lower alkoxy, lower alkoxycarbonyl, loweralkanoyl, lower alkylthio , lower alkyl sulfonyl, lower alkyl sulfinyl,perfluoro lower alkanoyl, nitro, or R₂₆ is a group of formula —NR₂₈R₂₉,

[0071] wherein

[0072] R₂₈ is H or lower alkyl,

[0073] R₂₉ is hydrogen, lower alkyl, lower alkoxycarbonyl, loweralkanoyl, aroyl, perfluoro lower alkanoylamino, lower alkyl sulfonyl,lower alkylaminocarbonyl, arylaminocarbonyl, or R₂₈ and R₂₉ takentogether form a 4, 5 or 6-membered saturated carbacyclic ring optionallycontaining one heteroatom selected from O, S, and N; with the carbonatoms in the ring being unsubstituted or substituted by lower alkyl orhalogen,

[0074] Q is —(CH₂)_(f)O—, —(CH₂)_(f)S—, —(CH₂)_(f)N(R₂₇)—, or—(CH₂)_(f)—,

[0075] R₂₇ is H, lower alkyl, aryl, lower alkanoyl, aroyl or loweralkoxycarbonyl,

[0076] e is an integer from 0 to 4, and

[0077] f is an integer from 0 to 3; and the dotted bond is optionallyhydrogenated;

[0078] and pharmaceutically acceptable salts and esters thereof.

[0079] Preferred compounds are as follows:

[0080] Compounds where X is a group of the formula

[0081] and Y, R₁₅ and R₁₆ are as in formula 1.

[0082] Such compounds where R₁₅ is lower alkyl, nitro, halogen,perfluoromethyl, or cyano and R₁₆ is hydrogen, lower alkyl, nitro,halogen, perfluoromethyl, or cyano, especially where R₁₅ and R₁₆ areindependently chloro or fluoro are preferred, especially where X-1 isselected from the group of

[0083] Compounds of formula 1 wherein X is a group of the formula X-2

[0084] and p, Y, R₁₅, R16, and R₃₀ are as in formula 1 (compound A)especially where Het is a 5- or 6-membered monocyclic heteroaromaticring containing 1, 2 or 3 nitrogens, or a nitrogen and a sulfur, or anitrogen and an oxygen or where Het is a bicyclic heteroaromatic ringcontaining from 1 to 3 nitrogens or where R₁₅ is nitro, lower alkylsulfonyl, cyano, lower alkyl, lower alkoxy, perfluorolower alkyl, loweralkylthio, lower alkanoyl, or aryl, especially where aryl isunsubstituted phenyl. In compound A R₁₆ may be hydrogen, halogen, nitro,cyano, lower alkyl, perfluoro lower alkyl; and R₃₀ is hydrogen or loweralkyl, or in compound A Het may be a 6 membered monocyclicheteroaromatic ring containing 1 or 2 nitrogens or a 10 memberedbicyclic heteroaromatic ring containing one nitrogen, R₁₅ is loweralkyl, or perfluoroalkyl and R₁₆ is hydrogen, lower alkyl, orperfluoroalkyl, and R₃₀ is absent. In compound A, X-2 may be selectedfrom the group of

[0085] Compounds of formula 1 wherein X is a group of formula X-3

[0086] and Y, R₁₈, R₁₉, and R₂₀ are as in formula 1 (compound B). Incompound B, it is preferred that R₁₈ is phenyl. In compound B it is alsopreferred that R₁₉ is lower alkyl which is unsubstituted or substitutedby pyridyl or phenyl. In compound B it is also preferred that R₂₀ issubstituted or unsubsituted lower alkanoyl. In compound B it is alsopreferred that R₁₈ is phenyl, R₁₉ is lower alkyl which is unsubstitutedor substituted by pyridyl or phenyl and R₂₀ is lower alkanoyl. Incompound B it is preferred that R₁₈ is phenyl which is unsubstituted orsubstituted by halogen or lower alkoxy; R₁₉ is phenyl lower alkyl whichis unsubstituted or substituted by lower alkoxy, pyridyl lower alkyl, orlower alkyl; and R₂₀ is substituted or unsubstituted lower alkanoyl.

[0087] In this latter compound, it is preferred that X-3 is selectedfrom the group of

[0088] Compounds of formula 1 where Y is a group of formula

[0089] and X, R₂₂, R₂₃, and R₂₄ are as in formula 1 (compound C). It ispreferred for compound C that R₂₂ and R₂₃ are lower alkyl,trifluoromethyl, or halogen and R₂₄ is hydrogen, lower alkyl, loweralkoxy, or halogen, especially when Y-1 is selected from the group of

[0090] Compounds of formula 1 wherein Yis a group of the formula Y-2

[0091] and p, X, Het, R₃₀ and R₃₁, are as in formula 1 (compound D). Itis preferred for compound D that Het is a 6 membered heteroaromaticring, especially where the heteroatom is N, and preferably where Y-2 isselected from the group of

[0092] Compounds of formula 1 where Y is a group of formula Y-3

[0093] and Y, R₂₅ and Q are as in formula 1, and the dotted bond can beoptionally hydrogenated (compound E). It is preferred for compound Ethat Y-3 is selected from the group of

[0094] A compound of formula 1 wherein X is a group of the formula X-1and Y is a group of the formula Y-1.

[0095] A compound of formula 1 wherein X is a group of the formula X-1and Y is a group of the

[0096] formula Y-2.

[0097] A compound of formula 1 wherein X is a group of the formula X-1 Yis a group of the formula Y-3 wherein R₁₅, R₁₆, R₂₅ and Q are as above;and the dotted bond can be optionally hydrogenated.

[0098] A compound of formula 1 wherein X is a group of the formula X-2and Y is a group of the formula Y-1.

[0099] A compound of formula 1 wherein X is a group of the formula X-2and Y is a group of the formula Y-2

[0100] A compound of formula 1 wherein X is a group of the formula X-2and Y is a group of the

[0101] formula Y-3 wherein R₁₅, R₁₆, R₂₅, R₃₀, Q and p are as above andthe dotted bond can be optionally hydrogenated.

[0102] A compound of formula 1 where X is a group of the formula X-3 andY is a group of the formula Y-1.

[0103] A compound of formula 1 wherein X is a group of the formula X-3and Y is a group of the formula Y-2.

[0104] A compound of formula 1 wherein X is a group of the formula X-3and Y is a group of the formula Y-3 where R₁₈, R₁₉, R₂₀, R₂₅, and Q areas above and the dotted bond can be optionally hydrogenated.

[0105] A compound of claim 1 wherein X is a group of the formula X-1

[0106] wherein R₁₆ is in the ortho position and is hydrogen, loweralkyl, nitro, cyano, halogen, lower alkylthio, perfluoroloweralkyl andR₁₅ is lower alkyl, nitro, cyano, halogen, lower alkylsulfonyl,perfluoroloweralkyl, and Y is a group of the formula Y-1

[0107] where R₂₂ is hydrogen, halogen, trifluoroalkyl, or lower alkyland R₂₃ is halogen, trifluoroalkyl, or lower alkyl, and R24 is hydrogenor Y is a group of the formula Y-3

[0108] wherein Q is as above and the dotted bond can be optionallyhydrogenated; R₂₅, is R₂₆—(CH₂)e—; e is 2-4 and R₂₆ is azido, cyano,hydroxy, lower alkoxy, lower alkoxycarbonyl, lower alkanoyl, lower alkylsulfonyl, lower alkyl sulfinyl, perfluoro lower alkanoyl, nitro, orlower alkylthio or R₂₅ is NHR₂₉ where R₂₉ is lower alkanoyl or loweralkylamino carbonyl (compound E).

[0109] In compound E it is preferred that X is a group of the formulaX-1

[0110] wherein R₁₆ is in the ortho position and is hydrogen, loweralkyl, nitro, cyano, halogen, lower alkylthio, perfluoroloweralkyl andR₁₅ is lower alkyl, nitro, cyano, halogen, lower alkylsulfonyl,perfluoroloweralkyl; and Y is a group of the formula Y-1

[0111] where R₂₂ is hydrogen, halogen, or lower alkyl and R₂₃ is halogenor lower alkyl, and R₂₄ is hydrogen, especially where R₁₆ is hydrogen orhalogen and R₁₅ is halogen; R₂₂ is hydrogen, halogen, ethyl, or methyland R₂₃ is halogen, ethyl, or methyl, and additionally where R₁₆ is inthe ortho position and R₁₅ and R₁₆ are both chlorine, and R₂₂ is methyland R₂₃ is chlorine or ethyl. An example of such a compound is4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-chloro-6-methylphenyl)thioxomethyl]-L-phenylalanine.

[0112] In compound E it is also preferred that X is a group of theformula X-1

[0113] wherein R₁₆ is in the ortho position and is hydrogen, loweralkyl, nitro, cyano, halogen, lower alkylthio, perfluoroloweralkyl andR₁₅ is lower alkyl, nitro, cyano, halogen, lower alkylsulfonyl,perfluoroloweralkyl; and Y is a group of the formula Y-3

[0114] which is a four to six membered cycloalkyl ring, R₂₅ isR₂₆-(CH₂)e—; e is 2-4 and R₂₆ is azido, cyano, hydroxy, lower alkoxy,lower alkoxycarbonyl, lower alkanoyl, lower alkyl sulfonyl, lower alkylsulfinyl, perfluoro lower alkanoyl, nitro, or lower alkylthio; and thedotted bond is hydrogenated. In such a compound it is preferred that R₁₆is hydrogen or halogen and R₁₅ is halogen; and Y-3 is a four or fivemembered ring and R₂₆ is lower alkoxy, lower alkyl sulfonyl, lower alkylsulfinyl, or lower alkylthio, especially where R₁₆ is in the orthoposition and R₁₅ and R₁₆ are both chlorine, and R₂₆ is lower alkylsulfonyl or lower alkylthio. An example of such a compound is4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[(4-methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine.

[0115] For another preferred compound E X is a group of the formula X-1

[0116] where R₁₆ is hydrogen or halogen and R₁₅ is halogen and Y is agroup of the formula Y-1

[0117] where R₂₂ is hydrogen, halogen, ethyl, or methyl and R₂₃ ishalogen, ethyl, or methyl and R₂₄ is hydrogen or Y is a group of theformula Y-3

[0118] where Y-3 is a four or five membered ring, R₂₅ is as in claim 39and R₂₆ is lower alkoxy, lower alkyl sulfonyl, lower alkyl sulfinyl, orlower alkylthio, and the dotted bond is optionally hydrogenated. Forsuch a compound it is preferred that R₁₆ is in the ortho position andR₁₅ and R₁₆ are both chlorine, and when Y is Y-1 then R₂₂ is methyl andR₂₃ is chlorine or ethyl and when Y is Y-3, Y-3 is a four or fivemembered ring and R₂₆ is lower alkyl sulfonyl or lower alkylthio.

[0119] A compound of formula 1 wherein Y is as in formula 1 and X is X-1

[0120] where R₁₅ is ortho and is halogen, lower alkyl, or perfluoroalkyland R₁₆ is hydrogen, halogen, lower alkyl, or perfluoroalkyl (compoundF).

[0121] For compound F it is preferred that R₁₅ is chlorine and R₁₆ ishydrogen or chlorine.

[0122] A compound of formula 1 wherein Y is as in formula 1 and X is X-2where Het is pyridine or pyrimidine and R₁₅ is lower alkyl orperfluoroalkyl R₁₆, and R₂₀ are hydrogen, lower alkyl, or perfluoroalkyl(compound G).

[0123] A compound of formula 1 wherein Y is as in formula 1 and X is X-3where R₁₉ is pyridinyl lower alkyl or phenyl lower alkyl, R₂₀ is loweralkanoyl, and R18 is phenyl (compound H).

[0124] A compound of formula 1 where X is as in formula 1 and Y is Y-1where R₂₂ is hydrogen or lower alkyl, R₂₃ is halogen, lower alkyl, orperfluoroalkyl, and R₂₄ is hydrogen, especially where R₂₂ is hydrogen ormethyl and R₂₃ is halogen, ethyl, or trifluoromethyl.

[0125] A compound of formula 1 wherein X is as in formula 1 and Y is Y-3which is a four to six membered cycloalkyl ring, R₂₅ is R₂₆—(CH₂)e—, eis 2-4, and R₂₆ is alkoxy, lower alkyl sulfonyl, loweralkylthio, orNHR₂₉ where R₂₉ is loweralkoxycarbonyl or loweralkylaminocarbonyl, andthe dotted bond is hydrogenated. It is preferred that R₂₆ is methoxy,methyl sulfonyl, or methylthio.

[0126] A preferred compound F has Y is Y-1 where R₂₂ is hydrogen orlower alkyl, R₂₃ is halogen, lower alkyl, or perfluoroalkyl, and R₂₄ ishydrogen. It is preferred that R₁₅ is chlorine and R₁₆ is hydrogen orchlorine. Examples are

[0127]4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-bromophenyl)thioxomethyl]-L-phenylalanine;

[0128]4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]L-phenylalanine.

[0129]4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalanine.

[0130]4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalanine.

[0131] In a preferred compound G, Y is Y-1 where R₂₂ is hydrogen orlower alkyl, R₂₃ is halogen, lower alkyl, or perfluoroalkyl, and R₂₄ ishydrogen.

[0132] In a preferred compound H, Y is Y-1 where R₂₂ is hydrogen orlower alkyl, R₂₃ is halogen, lower alkyl, or perfluoroalkyl, and R₂₄ ishydrogen. An example of such a compound is4-[(2S,4R)-3-acetyl-2-phenyl-4-[(3-pyridinyl)methyl]-5-oxo-imidazolidin-1-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalanine.

[0133] In a preferred compound F, Y is Y-3 which is a four to sixmembered cycloalkyl ring, R₂₅

[0134] is R₂₆—(CH₂)e—, e is 2-4, and R₂₆ is alkoxy, lower alkylsulfonyl, loweralkylthio, or NHR₂₉ where R₂₉ is loweralkoxycarbonyl orloweralkylaminocarbonyl, and the dotted bond is hydrogenated. It ispreferred that R₁₅ is chlorine and R₁₆ is hydrogen or chlorine. Examplesof such compounds are

[0135]4-[[2,6-dichlorophenyl)carbonyl]amino-N-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxomethyl]-L-phenylalanine.

[0136][[1-[2-[[(methylamino)carbonyl]amino]ethyl]cyclopentyl]thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]L-phenylalanine.

[0137]4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-L-phenylalanine.

[0138]4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1-[(4-methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.

[0139]4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1-(3-methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylalanine.

[0140]4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1-(3-methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalanine.

[0141] It is preferred that R26 is methoxy, methyl sulfonyl, or methylthio, especially where R₁₅ is chlorine and R₁₆ is hydrogen or chlorine.

[0142] A preferred compound G wherein Y is Y-3 which is a four to sixmembered cycloalkyl ring, R₂₅, is R₂₆—(CH₂)e—, e is 2-4, and R₂₆ isalkoxy, lower alkyl sulfonyl, loweralkylthio, or NHR₂₉ where R₂₉ isloweralkoxycarbonyl or loweralkylaminocarbonyl, and the dotted bond ishydrogenated. Preferably R₂₆ is methoxy, methyl sulfonyl, or methylthio. Examples are

[0143]4-[(2,6-dimethyl-3-pyridinylcarbonyl)amino]-N-[[1-[(4-methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine.

[0144]4-[[[4-(trifluoromethyl)-5-pyrimidinyl]carbonyl]amino]-N-[[1-(4-methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.

[0145]4-[[(2,4-dimethyl-6-trifluoromethyl-3-pyridinyl)carbonyl]amino]-N-[[1-[(4-methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.

[0146] In a preferred compound H, Y is Y-3 which is a four to sixmembered cycloalkyl ring, R₂₅

[0147] is R₂₆—(CH₂)e—, e is 2-4, and R₂₆ is alkoxy, lower alkylsulfonyl, loweralkylthio, or NHR₂₉ where R₂₉ is loweralkoxycarbonyl orloweralkylaminocarbonyl, and the dotted bond is optionally hydrogenated,especially where R₂₆ is methoxy, methyl sulfonyl, or methyl thio.Examples are

[0148] 4-[(2S,4R)-3-acetyl-2-phenyl-4-[(3-phenyl)methyl]-5-oxo-imidazolidin-1-yl]-N-[[(4-methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine

[0149] 4-[(2R,4R)-3-acetyl-2-phenyl-4-[(3-phenyl)methyl]-5-oxo-imidazolidin-1-yl]-N-[[(4-methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine.

[0150] As used in this specification, the terms are defined as follows:

[0151] The term “halogen” means bromine, chlorine, fluorine, or iodine,and the term “halo” means a halogen substituent.

[0152] The term “perfluoro” means complete substitution of all hydrogenatoms with fluoro substituted, as in perfluoro lower alkyl,perfluoroloweralkanoyl and perfluoroalkanoylamino. An example istrifluoromethyl.

[0153] The term “lower alkyl”, alone or in combination (for example aspart of lower alkanoyl, below), means a straight-chain or branched-chainalkyl group containing a maximum of six carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, sec.butyl, isobutyl, tert.butyl,n-pentyl, n-hexyl and the like. Lower alkyl groups may be unsubstitutedor substituted by one or more groups selected independently fromcycloalkyl, nitro, aryloxy, aryl (preferably phenyl or pyridyl), hydroxy(lower alkylhydroxy or hydroxylower alkyl), halogen, cyano, lower alkoxy(alkoxy lower alkyl or lower alkyl alkoxy), lower alkanoyl, loweralkylthio (lower alkylthio lower alkyl) sulfinyl (lower alkyl sulfinyl),sulfinyl lower alkyl (lower alkyl sulfinyl lower alkyl) sulfonyl (loweralkyl sulfonyl), sulfonyl lower alkyl (lower alkyl sulfonyl lower alkyl)perfluoro (perfluoro lower alkyl) and substituted amino such asaminosulfonyl (lower alkyl aminosulfonyl) or aminocarbonyl (lower alkylaminocarbonyl). Examples of substituted lower alkyl groups include2-hydroxylethyl, 3-oxobutyl, cyanomethyl, and 2-nitropropyl. The term“lower alkylthio” means a lower alkyl group bonded through a divalentsulfur atom, for example, a methyl mercapto or a isopropyl mercaptogroup.

[0154] The term “cycloalkyl” means an unsubstituted or substituted 3- to7-membered carbacyclic ring. Substituents useful in accordance with thepresent invention are hydroxy, halogen, cyano, lower alkoxy, loweralkanoyl, lower alkyl, aroyl, lower alkylthio, lower alkyl sulfinyl,lower alkyl sulfonyl, aryl, heteroaryl and substituted amino.

[0155] The term “lower alkoxy” means a lower alkyl group as definedabove, bonded through an oxygen atom. Examples are methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, tert-butoxy and the like.

[0156] The term “lower alkenyl” means a nonaromatic partiallyunsaturated hydrocarbon chain containing at least one double bond, whichis preferably 1-10 and more preferably 1-6 carbons in length. The groupmay be unsubstituted, or substituted with conventional substituents,preferably fluoro. Examples are vinyl, allyl, dimethylallyl, butenyl,isobutenyl, pentenyl.

[0157] The term “aryl” means a mono- or bicylic aromatic group, such asphenyl or naphthyl, which is unsubstituted or substituted byconventional substituent groups. Preferred substituents are lower alkyl,lower alkoxy, hydroxy lower alkyl, hydroxy, hydroxyalkoxy, halogen,lower alkylthio, lower alkylsulfinyl, lower alkylsulfonyl, cyano, nitro,perfluoroalkyl, alkanoyl, aroyl, aryl alkynyl, lower alkynyl,aminoalkylcarbonyl (arylaminocarbonyl) and lower alkanoylamino. Theespecially preferred substituents are lower alkyl, hydroxy, andperfluoro lower alkyl. Examples of aryl groups that may be used inaccordance with this invention are phenyl, p-tolyl, p-methoxyphenyl,p-chlorophenyl, m-hydroxy phenyl, m-methylthiophenyl,2-methyl-5-nitrophenyl, 2,6-dichlorophenyl, 1-naphthyl and the like.

[0158] The term “arylalkyl” means a lower alkyl group as hereinbeforedefined in which one or more hydrogen atoms is/are replaced by an arylgroup as herein defined. Any conventional aralkyl may be used inaccordance with this invention, such as benzyl and the like. Similarly,the term “heteroarylalkyl” is the same as an arylalkyl group except thatthere is a heteroaryl group as defined below in place of an aryl group.Either of these groups may be unsubstituted, or may be substituted onthe ring portion with conventional substituents such as

[0159] The term “heteroaryl” means an unsubstituted or substituted 5- or6-membered monocyclic hetereoaromatic ring or a 9- or 10-memberedbicyclic hetereoaromatic ring containing 1, 2, 3 or 4 hetereoatoms whichare independently N, S or O. Examples of hetereoaryl rings are pyridine,benzimidazole, indole, imidazole, thiophene, isoquinoline, quinzolineand the like. Substituents as defined above for “aryl” apply equallyhere in the definition of heteroaryl. The term “heteroaromatic ring” maybe used interchangeably with the term heteroaryl.

[0160] The term “lower alkoxycarbonyl” means a lower alkoxy group bondedvia a carbonyl group. Examples of alkoxycarbonyl groups areethoxycarbonyl and the like.

[0161] The term “lower alkylcarbonyloxy” means lower alkylcarbonyloxygroups bonded via an oxygen atom, for example an acetoxy group.

[0162] The term “lower alkanoyl” means lower alkyl groups bonded via acarbonyl group and embraces in the sense of the foregoing definitiongroups such as acetyl, propionyl and the like. Lower alkanoyl groups maybe unsubstituted, or substituted with conventional substituents such asalkoxy, lower alkyl, hydroxy, aryl, and hetereoaryl.

[0163] The term “lower alkylcarbonylamino” means lower alkylcarbonylgroups bonded via a nitrogen atom, such as acetylamino.

[0164] The term “aroyl” means an mono- or bicyclic aryl or heteroarylgroup bonded via a carbonyl group. Examples of aroyl groups are benzoyl,3-cyanobenzoyl, 2-naphthyl and the like. Aroyl groups may beunsubstituted, or substituted with conventional substituents such as

[0165] The term “aryloxy” means an aryl group, as hereinbefore defined,which is bonded via an oxygen atom. The preferred aryloxy group isphenoxy.

[0166] The term “electron-deficient substituent” means a substituent onan aromatic or heteroaromatic ring which has a positive Hammett sigmavalus as defined for example in Jerry March, Advanced Organic Chemistry,2^(nd) Edition, McGraw Hill, 1977, page 246-253. Typical electronwithdrawing groups are cyano, nitro, chloro, alkoxycarbonyl lower alkylsulfonyl, and aminocarbonyl.

[0167] In the compound of formula 1, Y is preferably the group Y-1whereby the invention comprises a compound of the formula:

[0168] wherein X, R₂₂, R₂₃ and R₂₄ are as above.

[0169] In the group Y-1, R₂₂ and R₂₃ are preferably lower alkyl orhalogen and R24 is preferably hydrogen.

[0170] Among the groups Y-1, when R₂₃ is lower-alkyl or halogen, Y-1 ispreferably:

[0171] When Y is a group Y-2, Y is preferably:

[0172] When Y is a group Y-3, Y is preferably:

[0173] The especially preferred groups X-1 are of the formula:

[0174] The especially preferred groups X-2 are of the formula:

[0175] The especially preferred groups X-3 are of the formula:

[0176] The compounds of the invention can exist as stereoisomers anddiastereomers, all of which are encompassed within the scope of thepresent invention.

[0177] The compounds of the invention inhibit the binding of VCAM-1 andfibronectin to VLA-4 on circulating lymphocytes, eosinophils, basophils,and monocytes (“VLA-4-expressing cells”). The binding of VCAM-1 andfibronectin to VLA-4 on such cells is known to be implicated in certaindisease states, such as rheumatoid arthritis, multiple sclerosis,inflammatory bowel disease, and particularly in the binding ofeosinophils to pulmonary endothelium which contributes to the cause ofthe pulmonary inflammation which occurs in asthma. Thus, the compoundsof the present invention would be useful for the treatment of asthma.

[0178] On the basis of their capability of inhibiting binding of VCAM-1and fibronectin to VLA-4 on circulating lymphocytes, eosinophils,basophils, and monocytes, the compounds of the invention can be used asmedicament for the treatment of disorders which are known to beassociated with such binding. Examples of such disorders are rheumatoidarthritis, multiple sclerosis, asthma, and inflammatory bowel disease.The compounds of the invention are preferably used in the treatment ofdiseases which involve pulmonary inflammation, such as asthma. Thepulmonary inflammation, which occurs in asthma, is related to eosinophilinfiltration into the lungs wherein the eosinophils bind to endotheliumwhich has been activated by some asthma-triggering event or substance.

[0179] Furthermore, compounds of the invention also inhibit the bindingof VCAM-1 and MadCAM to the cellular receptor alpha4-beta7, also knownas LPAM, which is expressed on lymphocytes, eosinophils and T-cells.While the precise role of alpha4-beta7 interaction with various ligandsin inflammatory conditions such as asthma is not completely understood,compounds of the invention which inhibit both alpha4-beta7 andalpha4-beta7 receptor binding are particularly effective in animalmodels of asthma. Furthermore work with monoclonal antibodies toalpha4-beta7 indicate that compounds which inhibit alpha4-beta7 bindingto MadCAM or VCAM are useful for the treatment of inflammatory boweldisease. They would also be useful in the treatment of other diseases inwhich such binding is implicated as a cause of disease damage orsymptoms.

[0180] The compounds of the invention can be administered orally,rectally, or parentally, e.g., intravenously, intramuscularly,subcutaneously, intrathecally or transdermally; or sublingually, or asopthalmalogical preparations, or as an aerosol in the case of pulmonaryinflammation. Capsules, tablets, suspensions or solutions for oraladministration, suppositories, injection solutions, eye drops, salves orspray solutions are examples of administration forms.

[0181] Intravenous, intramuscular, oral or inhalation administration isa preferred form of use. The dosages in which the compounds of theinvention are administered in effective amounts depending on the natureof the specific active ingredient, the age and the requirements of thepatient and the mode of administration. Dosages may be determined by anyconventional means, e.g., by dose-limiting clinical trials. Thus, theinvention further comprises a method of treating a host suffering from adisease in which VCAM-1 of fibronectin binding to VLA-4-expressing cellsis a causative factor in the disease symptoms or damage by administeringan amount of a compound of the invention sufficient to inhibit VCAM-1 orfibronectin binding to VLA-4-expressing cells so that said symptoms orsaid damage is reduced. In general, dosages of about 0.1-100 mg/kg bodyweight per day are preferred, with dosages of 1-25 mg/kg per day beingparticularly preferred, and dosages of 1- 10 mg/kg body weight per daybeing especially preferred.

[0182] The invention further comprises pharmaceutical compositions whichcontain a pharmaceutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier. Such compositionsmay be formulated by any conventional means. Tablets or granulates cancontain a series of binders, fillers, carriers or diluents. Liquidcompositions can be, for example, in the form of a sterilewater-miscible solution. Capsules can contain a filler or thickener inaddition to the active ingredient. Furthermore, flavor-improvingadditives as well as substances usually used as preserving, stabilizing,moisture-retaining and emulsifying agents as well as salts for varyingthe osmotic pressure, buffers and other additives can also be present.

[0183] The previously mentioned carrier materials and diluents cancomprise any conventional pharmaceutically acceptable organic orinorganic substances, e.g., water, gelatin, lactose, starch, magnesiumstearate, talc, gum arabic, polyalkylene glycols and the like.

[0184] Oral unit dosage forms, such as tablets and capsules, preferablycontain from 25 mg to 1000 mg of a compound of the invention.

[0185] The compounds of the present invention may be prepared by anyconventional means. In reaction Scheme 1, a 4-nitro-L-phenylalaninederivative of formula 1 in which R₁ is lower alkyl, which is a knowncompound or readily prepared by conventional means, is acylated with abenzoic acid derivative of formula 2 in which R₂ hydrogen, lower alkyl,lower alkoxy, cycloalkyl, aryl, arylalkyl, nitro, cyano, loweralkylthio, lower alkylsulfinyl, lower alkyl sulfonyl, lower alkanoyl,halogen, or perfluorolower alkyl, R₃ is hydrogen, halogen or lower alkyland R₄ is hydrogen, lower alkyl, lower alkoxy, aryl, nitro, cyano, loweralkyl sulfonyl, or halogen, using conventional means for amide bondformation. For example, a compound of formula 2 may be converted to thecorresponding acid chloride and condensed with a compound of formula 1in the presence of a proton acceptor such as a tertiary alkylamine.Alternatively compound 1 can be coupled with a carboxylic acid offormula 2 using standard peptide coupling conditions, for example HBTUin the presence of DIPEA in a polar, aprotic solvent such as DMF at atemperature between 0° C. and room temperature to give a compound offormula 3.

[0186] Conversion of the compound of formula 3 to the correspondingthioamide of formula 4 can be carried out by treatment with Lawesson'sreagent which is[2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide].The procedure is standard and has been described in detail. See forexample, Scheibey, S., Pedersen, B. S., Lawesson, S.-O. Bull Soc. Chim.Belg. 1978 87, 229 and Cava, M. P., Levinson, M. I., Tetrahedron 1985,41, 5061. The nitro group of the compound of formula 4 may be reduced tothe corresponding amine by any of the conventional means which arecompatible with thioamides. One convenient procedure employs zinc dustas the reducing agent in the presence of methanol, ammonium chloride andwater at a temperature of from 35 to 60° C. to give a compound offormula 5. Acylation of this compound with an aryl- or heteroarylcarboxylic acid of formula 6 using standard peptide coupling conditions,for example HBTU in the presence of DIPEA in a polar, aprotic solventsuch as DMF at a temperature between 0° C. and room temperature gives acompound of formula 7. In certain cases, for example with hinderedcarboxylic acids 6, it may be advantageous to form the correspondingacid halide and react it with the amine of formula 5, typically in thepresence of a slight excess of a base such as a tertiary amine or4-(dimethylamino)pyridine. The carboxylic acid of formula 6 may besubstituted by halogen, nitro, lower alkyl sulfonyl, cyano, lower alkyl,lower alkoxy, lower alkoxycarbonyl, carboxy, lower alkyl aminosulfonyl,perfluorolower alkyl, lower alkylthio, hydroxy lower alkyl, alkoxy loweralkyl, alkylthio lower alkyl, alkylsulfinyl lower alkyl, alkylsufonyllower alkyl, lower alkylsulfinyl, lower alkanoyl, aroyl, aryl, aryloxy.Where appropriate, it may also incorporate suitably protected reactivefunctionalities which must be removed to permit final conversion intocompounds of the invention. The choice and use of such groups will beapparent to those skilled in the art. Guidance for the selection and useof protecting groups is provided in standard reference works, forexample: “T. W. Green and P. G. M. Wuts, Protective Groups in OrganicSynthesis, 2nd edition, Wiley Interscience, New York, 1991. The estermoiety of compound 7 can generally be cleaved to the correspondingcarboxylic acid by treatment with an alkali metal hdyroxide, forexample, lithium hydroxide in aqueous methanol at a temperature of fromroom temperature to 50° C. Depending on the nature of R,, alternativeprocedures may be preferred. The choice of conditions for ester cleavagein the presence of functionalities such as thioamides is well known tothose skilled in the art.

[0187] Ortho-substituted benzoic acid derivatives which are notcommercially available can be prepared by conventional means. Forexample ortho-substituted aryl iodides or triflates may be carbonylatedin the presence of carbon monoxide and a suitable palladium catalyst.The preparation of such iodide or triflate intermediates is dependent onthe particular substitution pattern desired and they may be obtained bydirect iodination or diazotization of an aniline followed by treatmentwith a source of iodide for example, potassium iodide. Triflates may bederived from the corresponding phenols by conventional means such astreatment with trifluoromethane sulfonic anhydride in the presence of abase such as triethylamine or diisopropylethylamine in an inert solvent.Other means of obtaining ortho-substituted benzoic acids involvestreatment of an 2-methoxyphenyloxazoline derivative such as 9 with analkyl Grignard reagent followed by hydrolysis of the oxazoline ringfollowing the general procedure described by Meyers, A. I., Gabel, R.,Mihelick, E. D, J. Org. Chem. 1978, 43, 1372-1379., to give an acid offormula 10. 2- or 2,6-Disubstituted benzonitriles also serve asconvenient precursors to the corresponsing benzoic acids. In the case ofhighly hindered nitrites, for example 2-chloro-6-methylbenzonitrile,conventional hydrolysis under acidic or basic conditions is difficultand better results are obtained by DIBAL reduction to the correspondingbenzaldehyde followed by oxidation using a sodium chlorite/hydroperoxideoxidizing reagent.

[0188] Employing essentially the same procedures described in Scheme 1,utilizing a heteroaromatic carboxylic acid in place of 2, one canprepare compounds of formula 11.

[0189] For the synthesis of analogues a branched chain or cycloalkylmoiety, a similar procedure to that described in scheme 1 can beemployed starting with the appropriate branched chain or cycloalkylcarboxylic acid of formula 12. In this case, R₆ represents is loweralkyl, unsubstituted or fluorine substituted lower alkenyl, or asubstituted lower alkyl group wherein the substituents may be chosenfrom aryl, heteroaryl, azido, cyano, hydroxy, lower alkoxy, loweralkoxycarbonyl, lower alkylthio, lower alkyl sulfonyl, perfluoro loweralkanoyl, nitro, or a protected amino group. The amine protecting groupmust be chosen to be compatible with the reagents needed to convertcarboxamides to thioamides. Carbamates, for example, thetert-butoxycarbonyl moiety are suitable. As appropriate, theseprotecting groups may be removed by conventional means later in thesynthesis and the resulting free amine can be further functionalizedutilizing standard methods. For example, the amine can be acylated bytreatment with the appropriate anhydride, isocyanate or acid halide.

[0190] The synthesis of imidazolidinones of formula 21 is described inreaction scheme 3. An aminophenylalanine derivative of structure 13 inwhich R₆ is aryl, heteroaryl, branched chain alkyl or derived from acompound of formula 12, and R₇ is lower alkyl, is coupled with aN-protected alpha-amino acid of formula 14, in which R₈ can be a naturalor unnatural, D- or L-α-amino acid side chain and R₉ is a nitrogenprotecting group of the type conventionally used in peptide chemistry,for example, a Fmoc group, using standard peptide coupling conditions,for example HBTU in the presence of DIPEA in a polar, aprotic solventsuch as DMF at a temperature between 0° C. and room temperature to givea compound of formula 15. Depending on the nature of protecting groupR₉, an appropriate deprotection method is employed to give compound offormula 16. In the case of the protecting group R₉ is Fmoc group, it maybe removed from 15 using standard base treatment well known to thosepracticing peptide chemistry, for example with piperidine in DMF, toafford an amine of formula 16. The compound 16 can then react with analdehyde 17, in which R₁₀ is lower alkyl, aryl, or aryl lower alkyl, inthe presence of a water scavenger such as 4Å molecular sieves in anappropriate solvent such as dichloromethane or THF at 25-60° C. to givean imine of formula 18. The imine 18 may then be treated with anacylating agent such as the acyl chloride of formula 19 in which R₁₁ canbe an alkyl or aryl group in the presence of a base such DIPEA or DBU inan appropriate solvent such as dichloromethane or THF at 25-60° C. togive an acyl imidazolidinone of formula 20. Alternatively, otherreactive acylating groups such as acid anhydrides or mixed anhydridesmay be employed in this reaction. Compound 20 may be converted to acompound of the invention by an appropriate hydrolysis procedure, forexample by hydrolysis by treatment with an alkali metal hydroxide, forexample sodium hydroxide in aqueous alcohol to give, afteracidification, a carboxylic acid of formula 21.

[0191] General Melting points were taken on a Thomas-Hoover apparatusand are uncorrected. Optical rotations were determined with aPerkin-Elmer model 241 polarimeter. ¹H-NMR spectra were recorded withVarian XL-200 and Unityplus 400 MHz spectrometers, usingtetramethylsilane (TMS) as internal standard. Electron impact (EI, 70ev) and fast atom bombardment (FAB) mass spectra were taken on VGAutospec or VG 70E-HF mass spectrometers. Silica gel used for columnchromatography was Mallinkrodt SiliCar 230-400 mesh silica gel for flashchromatography; columns were run under a 0-5 psi head of nitrogen toassist flow. Thin layer chromatograms were run on glass thin layerplates coated with silica gel as supplied by E. Merck (E. Merck #1.05719) and were visualized by viewing under 254 nm UV light in a viewbox, by exposure to I₂ vapor, or by spraying with either phosphomolybdicacid (PMA) in aqueous ethanol, or after exposure to Cl₂, with a4,4′-tetramethyldiaminodiphenylmethane reagent prepared according to E.Von Arx, M. Faupel and M Brugger, J Chromatography, 1976, 120, 224-228.

[0192] Reversed phase high pressure liquid chromatography (RP-HPLC) wascarried out using either a Waters Delta Prep 4000 employing a 3×30 cm,Waters Delta Pak 15 μM C-18 column at a flow of 40 mL/min employing agradient of acetonitrile:water (each containing 0.75% TFA) typicallyfrom 5 to 95% acetonitrile over 35-40 min or a Rainin HPLC employing a41.4×300 mm, 8 μM, Dynamax™ C-18 column at a flow of 49 mL/min and asimilar gradient of acetonitrile:water as noted above. HPLC conditionsare typically described in the format (5-95-35-214); this refers to alinear gradient of from 5% to 95% acetonitrile in water over 35 minwhile monitoring the effluent with a UV detector set to a wavelength of214 nM.

[0193] Methylene chloride (dichloromethane), 2-propanol, DMF, THF,toluene, hexane, ether, and methanol, were Fisher reagent grade and wereused without additional purification except as noted, acetonitrile wasFisher hplc grade and was used as is.

[0194] Definitions:

[0195] THF is tetrahydrofuran,

[0196] DMF is N,N-dimethylformamide,

[0197] HOBT is 1-hydroxybenzotriazole,

[0198] BOP is [(benzotriazole-1-yl)oxy]tris-(dimethylamino)phosphoniumhexafluorophosphate,

[0199] HATU is O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate

[0200] HBTU is O-benzotriazole-N,N,N′,N′,-tetramethyluroniumhexafluorophosphate,

[0201] DIPEA is diisopropylethylamine,

[0202] DMAP is 4-(N,N-dimethylamino)pyridine

[0203] DPPA is diphenylphosphoryl azide

[0204] DPPP is 1,3-bis(diphenylphosphino)propane

[0205] DBU is 1,8-diazabicyclo[5.4.0]undec-7-ene

[0206] NaH is sodium hydride

[0207] brine is saturated aqueous sodium chloride solution

[0208] TLC is thin layer chromatography

[0209] LDA is lithium diisopropylamide

[0210] BOP-Cl is bis(2-oxo-3-oxazolidinyl)phosphinic chloride

[0211] NMP is N-methyl pyrrolidinone

[0212] Lawesson's reagent is[2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide]

EXAMPLES Example 1N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester.

[0213]

[0214] To a solution of[[1-(2-methoxyethyl)cyclopentyl]carbonyl]-4-nitro-L-phenylalanine methylester (4.30 g, 11.4 mmol) in toluene (20 mL) was added Lawesson'sreagent (2.60 g, 6.27 mmol). The resultant mixture was warmed to 50° C.and stirred for 18 h. The reaction mixture was filtered through asintered glass funnel and the filtrate was concentrated in vacuo. Theresidue was by flash column chromatography over silica gel (hexane-ethylacetate, 9:1 then 8:1) to affordN-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester (2.44 g, 54%; 70% based on recovered starting material) asa light yellow oil. HR MS: Obs. mass, 395.1639. Calcd. mass, 395.1640(M+H).

Example 24-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-(methoxyethyl)-cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester.

[0215]

[0216] To a suspension ofN-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester (4.58 g, 11.6 mmol), zinc dust (7.50 g, 116 mmol) andammonium chloride (9.20 g, 174 mmol) in methanol (200 mL) was added H₂O(100 mL) slowly over 5 min. After stirring for 20 min, the reactionmixture was partitioned between ethyl acetate (400 mL) and sat. ammoniumchloride solution (150 mL).The separated aqueous layer wasback-extracted with ethyl acetate (3×100 mL) and the organic layers werecombined, dried over Na₂SO₄, filtered and concentrated in vacuo. Theresidual oil was dried under high vacuum for 2 h to give crude4-amino-N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester (4.5 g).

[0217] To a solution of the crude amine obtained above (3.40 g, ˜8.77mmol based on 94% purity) and diisopropylethylamine (1.70 mL, 9.65 mmol)in dichloromethane (15 mL) was added a solution of 2,6-dichlorobenzoylchloride (1.9 g, 9.21 mmol) in dichloromethane (5 mL). The resultantmixture was stirred overnight, then was concentrated in vacuo andtransferred to a separatory funnel containing ethyl acetate (150 mL) andwater (40 mL). The aqueous layer was separated and back extracted withethyl acetate (1×50 mL). The combined organic layer was washed with asat. solution of Na₂CO₃ followed by brine, dried over MgSO₄, filteredand concentrated in vacuo. The reaction product by silica gel flashcolumn chromatography (hexane-ethyl acetate, 3:1) to give4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester (4.50 g, 95%). HR MS: Obs. mass, 559.1201. Calcd. mass,559.1201 (M+Na).

Example 34-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-L-phenylalanine.

[0218]

[0219] To a solution of4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-L-phenylalanine methyl ester (4.00 g, 7.44mmol) in methanol (18 mL) was added a solution of NaOH (421 mg, 10.5mmol) in water (3 mL). The mixture was stirred for 2 h and thenacidified (pH ˜1-2) with 0.5N HCl. The reaction mixture was poured intoa separatory funnel containing ethyl acetate (150 mL) and water (25 mL).The separated aqueous layer was back-extracted with ethyl acetate (2×50mL). The combined organic layers was washed with brine, dried overMgSO₄, filtered and concentrated in vacuo. Purification of the residualmaterial by RP-HPLC (15-95% acetonitrile-water gradient over 25 min)provided4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-L-phenylalanine(3.05 g, 78%). HR MS: Obs. mass, 545.1043. Calcd. mass, 545.1045 (M+Na).

Example 4 1-(2-azidoethyl)cyclopentanecarboxylic acid.

[0220]

[0221] To an ice cold solution of diisopropylamine (56 mL, 0.396 mol) inTHF (85 mL) was added n-butyl lithium in hexane solution (240 mL, 1.6M,0.393 mol) over 20 min. The mixture was stirred at 0° C. for 30 min,cooled to a bath temperature of −65° C. and ethylcyclopentanecarboxylate (37.4 g, 0.263 mol) in THF (50 mL) was addedover 20 min. After 1 h, a solution of 1,2-dibromoethane (47 mL, 0.545mol) in THF (50 mL) was added, the mixture was held at −65 ° C. for 3 hand then was allowed to warm to room temperature overnight. After thereaction was quenched by addition of saturated ammonium chloridesolution (200 mL), the layers were separated and the aqueous layer wasextracted with ethyl acetate (100 mL). The combined extracts were washedwith 1:1 brine:water (250 mL) and were dried (Na₂SO₄). The solution wasfiltered, concentrated in vacuo then the residue was diluted withtoluene (100 mL) and concentrated. The dilution and concentration cyclewas repeated twice to give ethyl 1-(2-bromoethyl)cyclopentanecarboxylate(52.5 g).

[0222] A solution of the above bromide (52.5 g, 0.211 mol) and sodiumazide (54 g, 0.831 mol) in DMF (200 mL) was stirred at 50° C. for 5 hunder a nitrogen atmosphere and was filtered. The filtrate wasconcentrated to near dryness, diluted with ethyl acetate (500 mL),filtered and concentrated to give crude ethyl1-(2-azidoethyl)cyclopentanecarboxylate (40.9 g) as a brown oil. Thismaterial was combined with product from a previous run (total 63.5 g)and was purified by chromatography over 250 g of silica gel (5% ethylacetate in hexane) to give 50.3 g of product as a light brown oil.

[0223] The oil from above (50.3 g, 0.238 mol) was dissolved in THF (750mL) and methanol (375 mL) and a solution of LiOH hydrate (15 g, 0.357mol) in water (300 mL) was added. The resulting solution was stirred at40° C. overnight and concentrated. The residue was dissolved in 2 L ofwater containing 40 mL of 1N NaOH and was washed with hexane (1 L). Theaqueous layer was treated with 1N HCl (375 mL) and was extracted withdiethyl ether (2×1 L). The combined extracts were dried (Na₂SO₄),filtered and concentrated under reduced pressure to give1-(2-azidoethyl)cyclopentane carboxylic acid (37.5 g) as an amberliquid.

Example 5N-[[1-(2-azidoethyl)cyclopentyl]carbonyl]-4-nitro-L-phenylalanine methylester.

[0224]

[0225] A solution of 4-nitro-L-phenylalanine methyl ester hydrochloride(3.0 g, 11.5 mmol), 1-(2-azidoethyl)cyclopentane carboxylic acid (2.3 g,12.7 mmol) and BOP (5.34 g, 12.1 mmol) in dichloromethane (6 mL) and DMF(4 mL) was treated with diisopropylethylamine (4.2 mL, 24.2 mmol). Themixture was stirred overnight at which time TLC (1:1 hexane:ethylacetate) indicated no more starting material. The mixture was dilutedwith water, extracted with ethyl acetate. The extracts were washed withwater and brine, then were dried (Na₂SO₄), filtered and evaporated invacuo. The residue was purified by chromatography over silica gel (3:1hexane:ethyl acetate) to afford 4.26 g ofN-[[1-(2-azidoethyl)cyclopentyl]carbonyl]-4-nitro-L-phenylalanine methylester.

Example 6N-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]cyclopentyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester.

[0226]

[0227] a. A solution ofN-[[1-(2-azidoethyl)cyclopentyl]carbonyl]-4-nitro-L-phenylalanine methylester (1.92 g, 4.93 mmol) in THF (20 mL) was treated dropwise with a 1Msolution of trimethylphosphine in THF. After the addition was complete,the mixture was stirred for 20 min and water (0.17 mL) was added. Thereaction was stirred a further 2 h, then a little trifluoroacetic acidwas added and the mixture was dried over sodium sulfate andconcentrated.

[0228] b. To a solution ofN-[[1-(2-aminoethyl)cyclopentyl]carbonyl]-4-nitro-L-phenylalanine methylester trifluoroacetic acid salt (2.35 g, 4.93 mmol) in dioxane (25 mL)was added diisopropylethylamine (0.860 mL, 4.93 mmol) and di-tert-butyldicarbonate (1.08 g, 4.93 mmol). The resultant mixture was stirred for18 h. The reaction mixture was filtered through a sintered glass funneland the filtrate was concentrated in vacuo. Purification of the residualmaterial by silica gel flash column chromatography (hexane-ethyl acetate3:1) afforded N-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]cyclopentyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester (2.20 g, 95%). HR MS: Obs. mass, 464.2397. Calcd. mass,464.2397 (M+H).

Example 7N-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester.

[0229]

[0230] To a solution ofN-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]cyclopentyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester (1.00 g, 2.16 mmol) in toluene/dioxane (1:1, 10 mL) wasadded Lawesson's reagent (0.524 g, 1.29 mmol). The resultant mixture waswarmed to 50° C. and was stirred for 24 h. The reaction mixture wasfiltered through a sintered glass funnel and the filtrate wasconcentrated in vacuo. Purification of the crude product by silica gelflash column chromatography (hexane-ethyl acetate, 6:1 then 4:1),affordedN-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester (460 mg, 44%; 65% based on recovered starting material) asa light yellow oil. HR MS: Obs. mass, 478.2014. Calcd. mass, 478.2012(M−H).

Example 8N-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester.

[0231]

[0232] To a solution ofN-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]-cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester (1.26 g, 2.63 mmol) in dichloromethane (15 mL) was addeddropwise trifluoroacetic acid (7 mL) and the resultant mixture wasstirred for 2 h at room temperature. The reaction mixture wasconcentrated in vacuo to afford the trifluoroacetic acid salt of crudeN-[[1-(2-aminoethyl)-cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester as a yellow oil (1.4 g).

[0233] To a solution of the salt obtained above (1.4 g, ˜2.63 mmol) indichloromethane (10 mL) was added diisopropylethylamine (1.37 mL, 7.88mmol) and acetic anhydride (0.250 mL, 2.63 mmol). The resultant mixturewas stirred overnight. The reaction mixture was concentrated in vacuoand transferred to a separatory funnel containing ethyl acetate (100 mL)and water (40 mL). The aqueous layer was separated and back extractedwith ethyl acetate (1×50 mL). The combined organic layer was washed withbrine, dried over MgSO₄, filtered and concentrated in vacuo. Theresulting residue was purified by flash column chromatography(dichloromethane-acetone, 5:1) to furnishN-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester (743 mg, 67%). HR MS: Obs. mass, 422.1744. Calcd. mass,422.1750 (M+H).

Example 94-amino-N-[[1-[2-acetylamino)ethyl]cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester.

[0234]

[0235] To a suspension ofN-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester (740 mg, 1.75 mmol), zinc dust (1.14 g, 17.5 mmol) andammonium chloride (1.41 g, 26.3 mmol) in methanol (20 mL) was added H₂O(10 mL) slowly over 5 min. After stirring for 20 min, the reactionmixture was diluted with ethyl acetate (80 mL) and sat. ammoniumchloride solution (25 mL). The separated aqueous layer wasback-extracted with ethyl acetate (3×25 mL) and the organic layers werecombined, dried over Na₂SO₄, filtered and concentrated in vacuo. Theresidual oil was dried under high vacuum for 2 h to give the crude amine(750 mg)which was by flash silica gel column chromatography(dichloromethane-acetone 2:1) to furnish4-amino-N-[[1-[2-acetylamino)ethyl]cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester (650 mg, 95%). HR MS: Obs. mass, 392.2016. Calcd. mass,392.2008 (M+H).

Example 10 N-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalanine.

[0236]

[0237] To a solution of4-amino-N-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester (195 mg, 0.498 mmol) and diisopropylethylamine (0.0950 mL,0.548 mmol) in dichloromethane (1 mL) was added a solution of2,6-dichlorobenzoyl chloride (110 mg, 0.523 mmol) in dichloromethane (1mL). The resultant mixture was stirred overnight then was concentratedin vacuo and transferred to a separatory funnel containing ethyl acetate(50 mL) and water (10 mL). The separated aqueous layer back-extractedwith ethyl acetate (1×25 mL). The combined organic layer was washed inturn sat. solution of Na₂CO₃ and brine, then was dried over MgSO₄,filtered and concentrated in vacuo to provide crudeN-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalaninemethyl ester (300 mg).

[0238] To a solution of the above methyl ester (300 mg, ˜0.498 mmol) inmethanol (1 mL) was added a solution of NaOH (64 mg, 14.9 mmol) in water(1 mL). The mixture was stirred for 2 h and was then acidified (pH ˜1-2)with 0.5M HCl. The reaction mixture was poured into a separatory funnelcontaining ethyl acetate (50 mL) and water (10 mL) and the separatedaqueous layer was back-extracted with ethyl acetate (2×25 mL). Thecombined organic layers were washed with brine, dried over MgSO₄,filtered and concentrated in vacuo. Purification of the crude by RP-HPLC(15-95% acetonitrile-water gradient over 25 min) and lyophylization ofthe appropriate fractions, provided N-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalanine(126 mg, 46%) as a colorless solid. HR MS: Obs. mass, 550.1330. Calcd.mass, 550.1334 (M+H).

Example 114-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[[(methylamino)carbonyl]amino]ethyl]cyclopentyl]thioxomethyl]-L-phenylalanine.

[0239]

[0240]4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[2-[[(methylamino)carbonyl]amino]ethyl]cyclopentyl]thioxomethyl]-L-phenylalaninewas prepared fromN-[[1-[2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl]cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester and methyl isocyanate by using the general proceduredescribed in examples 8 to 10. HR MS: Obs. mass, 565.1436. Calcd. mass,565.1443 (M+H).

Example 12 2-chloro-6-methylbenzaldehyde.

[0241]

[0242] A 500 mL, three-necked, round bottomed flask equipped with amagnetic stirrer, thermometer, addition funnel, and argon inlet wascharged with 75 g (494 mmol) of 2-chloro-6-methylbenzonitrile and 400 mLof toluene (stored over 4 Å molecular sieves). The mixture was cooled to−2° C. (ice/acetone bath) and a 1M solution of DIBAL-H in hexanes (593mmol, 593 mL) was added dropwise over a period of 30 min whilemaintaining the temperature below 0° C. After the addition wascompleted, the reaction mixture was stirred for 1 h at 0° C. and thenallowed to warm to room temperature. After 2 h at room temperature, TLCanalysis indicated the absence of starting material (4:1 hexane:diethylether, phosphomolybdic acid spray, as analysis by UV fluorescence wasmisleading). The reaction was poured into a mixture of ice (2000 g) andconcentrated sulfuric acid (50 mL) and was stirred for overnight. Theprecipitated solids were collected by filtration and the filtrate wasextracted with diethyl ether (2×200 mL). The combined extracts werewashed with brine, dried (MgSO₄) and filtered. Evaporation of thesolvent under reduced pressure gave the crude aldehyde, which wascombined with the above solid to afford 71.31 g (93%) of light yellowcolored aldehyde suitable for use in the next step.

Example 13 2-chloro-6-methylbenzoic acid.

[0243]

[0244] A 1000 mL, three-necked, round bottomed flask equipped with amagnetic stirrer, thermometer, addition funnel, and argon inlet wascharged with 2-chloro-6-methylbenzaldehyde (71.31 g, 461 mmol, crudeobtained from the above experiment) and 750 mL of acetonitrile. To thissuspension, a solution of monobasic sodium phosphate (115 mmol, 15.9 g)in water 240 mL) was added followed by hydrogen peroxide (50 mL, 30%) atroom temperature. Then, a solution of sodium chlorite (73.5 g, 811 mmol)in water (700 mL) was added dropwise at 0° C. while maintaining thetemperature below 3° C. After the addition was complete, the yellowsuspension was stirred for 15 h at 0° C. to room temperature. TLCanalysis of the mixture indicated the absence of starting material. Asolution of sodium bisulfite (73 g, 701 mmol) in water (200 mL) wasadded dropwise at 0° C. until the yellow color disappeared (KI-paperpositive). Cooling was maintained throughout to control the exothermicreaction. The solvent was removed under vacuum to afford a colorlesssolid. The solid was collected by filtration and the filtrate wasextracted with diethyl ether (200 mL). The above solid was dissolved inthe combined diethyl ether extracts which were then washed with 10% NaOHsolution (2×200 mL). The combined aqueous washings were acidified with10% HCl to pH 1. The resulting colorless precipitate was collected byfiltration and air-dried to afford 54.88 g (65%, overall in two steps)of 2-chloro-6-methylbenzoic acid as a colorless solid.

Example 14 N-[(2-chloro-6-methylphenyl)carbonyl]-4-nitro-L-phenylalaninemethyl ester.

[0245]

[0246] To a solution of 4-nitro-L-phenylalanine methyl esterhydrochloride salt (7.44 mmol, 1.94 g), 2-chloro-6-methylbenzoic acid(8.2 mmol, 1.4 g) and HBTU (8.2 mmol, 3.11 g) in DMF (27 mL) was addeddiisopropylethylamine (18.6 mmol, 3.24 mL) at room temperature. Theclear solution was stirred for 48 h at room temperature and was dilutedwith 100 mL of ethyl acetate. The solution was washed in turn with 0.5Nhydrochloric acid (2×50 mL), saturated sodium bicarbonate solution (2×50mL), brine (100 mL) then was dried (MgSO₄) and filtered. Concentrationof the solution to dryness gave 2.67 g (95%) ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-nitro-L-phenylalanine methylester as a colorless solid, mp 120-123° C. HRMS: Obs. mass, 376.4274.Calcd. mass, 376.4238 (M+H).

Example 15N-[(2-chloro-6-methylphenyl)thioxomethyl]-4-nitro-L-phenylalanine methylester.

[0247]

[0248] To a mixture ofN-[(2-chloro-6-methylphenyl)carbonyl]-4-nitro-L-phenylalanine methylester (9.66 mmol, 3.64 g) and Lawesson's reagent (6.0 mmol, 2.46 g, 0.62equiv.) was added toluene (15 mL, which had been stored over 4 Åmolecular sieves) at room temperature. The suspension was heated to90-100° C. and was stirred for 24 h. Examination of the resulting clearsolution by TLC failed to detect the presence of starting material. Thereaction mixture was diluted with ethyl acetate (50 mL) and washed withwater (50 mL), saturated sodium bicarbonate solution (50 mL), and brine(50 mL). The organic extract was dried (MgSO₄) filtered and evaporatedunder reduced pressure. The crude compound was purified by carefulsilica gel column chromatography (hexane:ethyl acetate, 4:1 to 2:1) toobtain 1.52 g (40%) ofN-[(2-chloro-6-methylphenyl)thioxomethyl]-4-nitro-L-phenylalanine methylester as a yellow solid, mp 150-153° C. (triturated from diethylether/hexane 3:1 ratio). HRMS: Obs. mass, 393.0685. Calcd. mass,393.0677 (M+H).

Example 164-amino-N-[(2-chloro-6-methylphenyl)thioxomethyl]-L-phenylalanine methylester.

[0249]

[0250] To a mixture ofN-[(2-chloro-6-methylphenyl)thioxomethyl]-4-nitro-L-phenylalanine methylester (3.86 mmol, 1.52 g), zinc dust (˜325 mesh, 39.0 mmol, 2.55 g, 10equiv.) and ammonium chloride (58.0 mmol, 3.09 g, 15 equiv.) was addedmethanol (50 mL) and water (25 mL) at room temperature. After additionof water, an exothermic reaction ensued and the temperature rose tobetween 45 and 50° C. After the suspension was stirred for 2 h at a bathtemperature of 50-60° C., TLC analysis of the mixture indicated theabsence of starting material. The reaction mixture was filtered througha pad of celite and the filter cake was washed with methanol (50 mL) andwater (40 mL). The filtrate was concentrated under vacuum to removemethanol and the product was extracted into ethyl acetate (2×50 mL). Thecombined extracts were washed with brine (50 mL), dried (MgSO₄),filtered and concentrated in vacuo to give 1.3 g (92%) of4-amino-N-[(2-chloro-6-methylphenyl)thioxomethyl]-L-phenylalanine methylester as an amorphous yellow solid, which was used directly for nextstep. HRMS: Obs. mass, 363.0932. Calcd. mass, 363.0934 (M+H).

Example 17N-[(2-chloro-6-methylphenyl)thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalaninemethyl ester.

[0251]

[0252] To a solution of4-amino-N-[(2-chloro-6-methylphenyl)thioxomethyl]-L-phenylalanine methylester (3.57 mmol, 1.296 g) and 2,6-dichlorobenzoyl chloride (3.75 mmol,0.785 g) in dichloromethane (20 mL) was added diisopropylethylamine(5.35 mmol, 0.93 mL) at room temperature. The solution was stirred for15 h at which time TLC analysis of the mixture indicated the absence ofstarting material. After the addition of water (30 mL), the layers wereseparated and the aqueous phase was extracted with dichloromethane (20mL). The combined extracts were washed with brine (50 mL), dried (MgSO₄)and concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (hexane:ethyl acetate, 4:1 to 1:1) toobtain 1.91 g (83%) ofN-[(2-chloro-6-methylphenyl)thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalaninemethyl ester as an amorphous colorless solid. HRMS: Obs. mass, 535.0399.Calcd. mass, 535.0416 (M+H).

Example 184-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-chloro-6-methylphenyl)thioxomethyl]-L-phenylalanine.

[0253]

[0254] To a suspension ofN-[(2-chloro-6-methylphenyl)thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalaninemethyl ester (2.89 mmol, 1.55 g) in ethanol (8 mL) was added aqueous1.0N sodium hydroxide (5 mL) at room temperature. The mixture was heatedto 50-55° C. and the resulting clear solution was stirred for 3-4 h. TLCanalysis of the mixture indicated the absence of starting material. Themixture was concentrated to remove ethanol, then was diluted with 15 mLof water and extracted with 25 mL of diethyl ether to remove any neutralimpurities. The aqueous layer was acidified with 1N HCl and theprecipitated colorless solid was extracted into ethyl acetate (2×30 mL).The combined extracts were washed with brine, dried (MgSO₄) andconcentrated in vacuo to afford 1.45 g (96%) ofN-[(2-chloro-6-methylphenyl)thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalanineas an amorphous colorless solid. HRMS: Obs. mass, 521.0241. Calcd. mass,521.0260 (M+H).

Example 19N-[(2-chloro-6-methylphenyl)thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalaninesodium salt.

[0255]

[0256]N-[(2-chloro-6-methylphenyl)thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]-L-phenylalanine(2.77 mmol, 1.45 g) was dissolved in water (10 mL) containing 1.5equivalents of aqueous 1.0N sodium hydroxide (4.2 mL) at roomtemperature. The solution was loaded into a reverse phase column size of8 inches length with 1.5 inches diameter containing C-18 silica gel andeluted with water to remove excess base. The product was eluted with5-20% methanol in water. After the appropriate fractions were combinedand concentrated, the residue was dissolved in 50 mL water andlyophilized to afford 1.3 g of the sodium salt as a colorless amorphoussolid. HRMS: Obs. mass, 543.0076. Calcd. mass, 543.0079 (M+H).

Example 20 2-ethyl-6-methylbenzoic acid.

[0257]

[0258] A 250 mL pressure bottle was charged with2-ethyl-6-methyliodobenzene (30.07 mmol, 7.4 g), Pd(OAc)₂ (1.43 mmol,334 mg) and dppp (1.43 mmol, 620 mg). The flask was closed with a septumand evacuated three times with argon. Acetonitrile (96 mL),triethylamine (189 mmol, 19.0 g, 26.25 mL) and water (19.1 mL) wereadded in succession by the aid of syringe and the rubber septum wasreplaced with a teflon lined cap connected to a carbon monoxide source.The flask was now pressurized with carbon monoxide (40 psi) and theexcess pressure was released. This process was repeated three times andfinally the mixture was stirred for 5 min under 40 psi carbon monoxidepressure. The flask was then disconnected from the carbon monoxidecylinder and immersed in a preheated oil bath (83-85° C.). The reactionmixture turned black within 1 h and was stirred for another 14 h at thistemperature, then was cooled to room temperature and the pressure wasreleased. The resulting mixture was diluted with diethyl ether (200 mL)and 1.0N NaOH (20 mL). The formed sodium was extracted into water (2×100mL). The combined water extracts were acidified with 1.0N HCl and themixture was extracted with dichloromethane (3×100 mL). The combineddichloromethane extracts were washed with brine, dried (MgSO₄), filteredand the volatiles were removed under vacuum to provide 3.58 g (72.5%) of2-ethyl-6-methylbenzoic acid as a viscous brown oil which slowlysolidified overnight. HR MS: Obs. mass, 164.0833. Calcd. mass, 164.0837(M+).

Example 21 N-[(2-ethyl-6-methylphenyl)carbonyl]-4-nitro-L-phenylalaninemethyl ester.

[0259]

[0260] Using the procedure described in example 14,N-[(2-ethyl-6-methylphenyl)carbonyl]-4-nitro-L-phenylalanine methylester was prepared in 72% yield as a colorless solid, mp 119-121° C. HRMS: Obs. mass, 371.1610. Calcd. mass, 371.1607 (M+H).

Example 22N-[(2-ethyl-6-methylphenyl)thioxomethyl]-4-nitro-L-phenylalanine methylester.

[0261]

[0262] Using the procedure described in example 15,N-[(2-ethyl-6-methylphenyl)thioxomethyl]-4-nitro-L-phenylalanine methylester was prepared in 47% yield as an amorphous colorless solid. HR MS:Obs. mass, 387.1383. Calcd. mass, 387.1378 (M+H).

Example 234-amino-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalanine methylester.

[0263]

[0264] Using the general procedure described in example 16,4-amino-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalanine methylester was prepared in 94% yield as an amorphous colorless solid. HR MS:Obs. mass, 357.1640. Calcd. mass, 357.1638 (M+H).

Example 244-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalaninemethyl ester.

[0265]

[0266] Using the procedure described in example 17,4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalaninemethyl ester was prepared in 70% yield as an amorphous colorless solid.HR MS: Obs. mass, 529.1094. Calcd. mass, 529.1119 (M+H).

Example 254-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalanine.

[0267]

[0268] Using the procedure described in example 18,4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalaninewas prepared in 77% yield as an amorphous colorless solid. HR MS: Obs.mass, 515.0942. Calcd. mass, 515.0963 (M+H).

Example 26 N-[(2-ethyl-6-methylphenyl)thioxomethyl]-4-[[(2R)-2-(Fmoc-amino)-1-oxo-3-(pyridin-3-yl)propyl]amino]-L-phenylalaninemethyl ester.

[0269]

[0270] Using the procedure described in example 1,N-[(2-ethyl-6-methylphenyl)thioxomethyl]-4-[[(2R)-2-(Fmoc-amino)-1-oxo-3-(pyridin-3-yl)propyl]amino]-L-phenylalaninemethyl ester was prepared in 72% yield as an amorphous colorless solid.HR MS: Obs. mass, 727.2973. Calcd. mass, 727.2954 (M+H).

Example 274-[[(2R)-2-amino-1-oxo-3-(pyridin-3-yl)propyl]amino]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalaninemethyl ester.

[0271]

[0272]N-[(2-ethyl-6-methylphenyl)thioxomethyl]-4-[[(2R)-2-(Fmoc-amino)-1-oxo-3-(pyridin-3-yl)propyl]amino]-L-phenylalaninemethyl ester from example 26 (0.308 mmol, 224 mg) was treated with 25%piperidine in NMP (3 mL) and the solution was stirred at roomtemperature. Within 1 h, TLC analysis of the mixture indicated theabsence of starting material. The mixture was diluted with hexane (25mL) and the formed layers were separated. The bottom yellow layer waswashed with hexane, then was diluted with water and extracted with ethylacetate and THF (2:1, 3×25 mL). The combined extracts were washed withwater (50 mL), brine (50 mL), dried (MgSO₄), filtered and the solventswere removed under vacuum. The resulting residue was dried under highvacuum to afford 126 mg (81%) of4-[[(2R)-2-amino-1-oxo-3-(pyridin-3-yl)propyl]amino]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalaninemethyl ester as an amorphous colorless solid. HR MS: Obs. mass,505.2270. Calcd. mass, 505.2274 (M+H).

Example 284-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-[(pyridin-3-yl)methyl]imidazolidin-1-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalaninemethyl ester.

[0273]

[0274] To a solution of4-[[(2R)-2-amino-1-oxo-3-(pyridin-3-yl)propyl]amino]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalaninemethyl ester (0.224 mmol, 113 mg) in dichloromethane (0.75 mL) andtrimethyl orthoformate (0.75 mL), was added benzaldehyde (0.25 mmol,27.5 mg). After the resulting light yellow solution was stirred for 3days at room temperature, it was heated to 90° C. (oil bath temperature)and excess acetic anhydride (2.0 mmol, 0.21 mL) was introduced via asyringe. The solution was stirred at 110-120° C. (oil bath temperature)for 6 h, then was cooled to room temperature and the solvents wereremoved in vacuo. The crude residue was purified by RP-HPLC to obtain 95mg (67%) of4-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-[(pyridin-3-yl)methyl]imidazolidin-1-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalaninemethyl ester as an amorphous colorless solid. HR MS: Obs. mass,635.2672. Calcd. mass, 635.2692 (M+H). Another isomer was formed in veryminor amount by HPLC (<5%) and not attempted to isolate it.

Example 294-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-[(pyridin-3-yl)methyl]imidazolidin-1-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalanine.

[0275]

[0276] The hydrolysis of4-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-[(pyridin-3-yl)methyl]imidazolidin-1-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalaninemethyl ester was carried out using the general procedure described inexample 18. The obtained crude product was purified by RP-HPLC, using a5-95% acetonitrile-water gradient over 30 min and the appropriatefraction was collected. The acetonitrile was removed under vacuum andthe product was extracted into ethyl acetate:THF (3:1) (2×25 mL). Thecombined extracts were washed with brine, dried (MgSO₄), filtered andthe solvents were removed under reduced pressure. The resulting residuewas dried under high vacuum to obtain4-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-[(pyridin-3-yl)methyl]imidazolinidin-1-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalaninein 30% yield as an amorphous colorless solid. HR MS: Obs. mass,621.2520. Calcd. mass, 621.2535 (M+H).

Example 30 N-[(2-fluorophenyl)carbonyl]-4-nitro-L-phenylalanine methylester.

[0277]

[0278] Using the general procedure described in example 14, startingwith 4-nitro-L-phenylalanine methyl ester hydrochloride salt and2-fluorobenzoic acid,N-[(2-fluorophenyl)carbonyl]-4-nitro-L-phenylalanine methyl ester wasprepared in 99% yield as a colorless solid, mp 137-139° C. HR MS: Obs.mass, 346.0977. Calcd. mass, 346.0980, M+.

Example 31 N-[(2-fluorophenyl)thioxomethyl]-4-nitro-L-phenylalaninemethyl ester.

[0279]

[0280] Using the general procedure described in example 15, startingwith N-[(2-fluorophenyl)carbonyl]-4-nitro-L-phenylalanine methyl ester,N-[(2-fluorophenyl)thioxomethyl]-4-nitro-L-phenylalanine methyl esterwas prepared in 99% yield as an amorphous colorless solid. HR MS: Obs.mass, 363.0816. Calcd. mass, 363.0815 (M+H).

Example 32 4-amino-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalaninemethyl ester.

[0281]

[0282] Using the general procedure described in example 16, startingwith N-[(2-fluorophenyl)thioxomethyl]-4-nitro-L-phenylalanine methylester, 4-amino-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalanine methylester was prepared in 87% yield as an amorphous colorless solid. HR MS:Obs. mass, 332.1042. Calcd. mass, 332.1046, (M+H).

Example 334-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalaninemethyl ester.

[0283]

[0284] Using the general procedure described in example 17, startingwith 4-amino-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalanine methylester and 2,6-dichlorobenzoyl chloride,4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalaninemethyl ester was prepared in 74% yield as an amorphous colorless solid.HR MS: Obs. mass, 505.0561. Calcd. mass, 505.0555, (M+H).

Example 344-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalanine.

[0285]

[0286] Using the general procedure described in example 18, startingwith4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalaninemethyl ester,4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalaninewas prepared in 89% yield as an amorphous colorless solid. HR MS: Obs.mass, 491.0407. Calcd. mass, 491.0399 (M+H).

Example 354-nitro-N-[[(2-(trifluoromethyl)phenyl]carbonyl]-L-phenylalanine methylester.

[0287]

[0288] Using the general procedure described in example 14, startingwith 4-nitro-L-phenylalanine methyl ester hydrochloride salt and2-trifluoromethylbenzoic acid,4-nitro-N-[[(2-(trifluoromethyl)phenyl]carbonyl]-L-phenylalanine methylester was prepared in 69% yield as a colorless solid, mp 152-154° C. HRMS: Obs. mass, 397.1017. Calcd. mass, 397.1011 (M+H).

Example 364-nitro-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalaninemethyl ester

[0289]

[0290] Using the general procedure described in example 15, startingwith 4-nitro-N-[[(2-(trifluoromethyl)phenyl]carbonyl]-L-phenylalaninemethyl ester,4-nitro-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalaninemethyl ester was prepared in 67% yield as an amorphous colorless solid.HR MS: Obs. mass, 412.0752. Calcd. mass, 412.0757 (M+H).

Example 374-amino-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalaninemethyl ester.

[0291]

[0292] Using the general procedure described in example 16, startingwith 4-nitro-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalaninemethyl ester,4-amino-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalaninemethyl ester was prepared in 98% yield as an amorphous colorless solid.HR MS: Obs. mass, 382.1072. Calcd. mass, 382.1078, (M+H).

Example 384-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalanine methyl ester.

[0293]

[0294] Using the general procedure described in example 17, startingwith 4-amino-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalaninemethyl ester and 2,6-dichlorobenzoyl chloride,4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)phenyl]-thioxomethyl]-L-phenylalaninemethyl ester was prepared in 98% yield as an amorphous colorless solid.HR MS: Obs. mass, 555.0511. Calcd. mass, 555.0524, (M+H).

Example 394-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalanine.

[0295]

[0296] Using the general procedure described in example 18, startingwith 4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalanine methyl ester,4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalaninewas prepared in 99% yield as an amorphous colorless solid. HR MS: Obs.mass, 541.0358. Calcd. mass, 541.0367, (M+H).

Example 40 1-(4-bromobutyl)cyclopentanecarboxylic acid methyl ester.

[0297]

[0298] To a solution of diisopropylamine (150 mmol, 21 mL) in THF (100mL) at −10 ° C. was added dropwise a 2.5M solution of n-butyl lithium inhexanes (145 mmol, 58 mL) while maintaining the temperature below 0° C.After the addition was complete, the solution was stirred for 30 min at0° C., then it was cooled to −70° C. using a dry ice/acetone bath. Asolution of methyl cyclopentanecarboxylate (100 mmol, 13.1 g) in THF (20mL) was added dropwise at −70° C. maintaining the reaction temperaturebetween −60 to −70° C. The mixture was then stirred for 1 h at −50 to−60° C. and a solution of 1,4-dibromobutane (100 mmol, 21.59 g) in THF(20 mL) was added dropwise and the light brown suspension was stirredfor 1 h at −60 to −70° C. The cooling bath was removed and the reactionwas allowed to equilibrate to room temperature and stirred overnight.The reaction mixture then was poured into a saturated solution ofammonium chloride (200 mL) and the mixture was extracted with diethylether (2×100 mL). The combined extracts were washed with brine (150 mL),dried (MgSO₄), filtered and the solution was concentrated under reducedpressure. The resulting residue was distilled at 120-133° C./2.5 mm Hgto obtain 12.8 g (48%) of 1-(4-bromobutyl)cyclopentanecarboxylic acidmethyl ester as a colorless oil. HR MS: Obs. mass, 262.0565. Calcd.mass, 262.0568, (M+).

Example 41 1-[4-(methylthio)butyl]cyclopentanecarboxylic acid methylester.

[0299]

[0300] To a solution of 1-(4-bromobutyl)cyclopentanecarboxylic acidmethyl ester (38 mmol, 10 g) in DMF (100 mL) was added sodiumthiomethoxide (72.6 mmol, 5.09 g). After the addition, an exothermicreaction ensued and the mixture turned to a light brown cloudy solution.The mixture was stirred for 15 h at room temperature, then was pouredinto water (200 mL) and extracted with diethyl ether (2×150 mL). Thecombined extracts were washed with brine (150 mL), dried (MgSO₄),filtered and the solution was concentrated in vacuo. The residualmaterial was purified by silica gel column chromatography to afford 4.43g (51 %) of methyl 1-[4-(methylthio)butyl]cyclopentanecarboxylic acidmethyl ester as a colorless oil. HR MS: Obs. mass, 230.1343. Calcd.mass, 230.1341, (M+).

Example 42 1-[4-(methylsulfonyl)butyl]cyclopentanecarboxylic acid methylester.

[0301]

[0302] To a solution of 1-[4-(methylthio)butyl]cyclopentanecarboxylicacid methyl ester (19.2 mmol, 4.43 g) in acetic acid (20 mL) was added30% hydrogen peroxide (10 mL) and stirred mixture was heated to 70° C.for 15 h. TLC analysis of the mixture indicated the absence of startingmaterial. The reaction mixture was cooled to room temperature and wasconcentrated under vacuum. The residue was poured into saturated sodiumbicarbonate solution and was extracted with diethyl ether (3×100 mL).The combined extracts were washed with brine (200 mL), dried (MgSO₄),filtered and the solvent was removed under reduced pressure. Theresidual material was purified by silica gel column chromatography toafford 4.94 g (98%) of 1-[4-(methylsulfonyl)butyl]cyclopentanecarboxylicacid methyl ester as a colorless oil. LR MS (C12H22O4S): 263 (M+H).

Example 43 1-[4-(methylsulfonyl)butyl]cyclopentane carboxylic acid

[0303]

[0304] To a solution of1-[4-(methylsulfonyl)butyl]cyclopentanecarboxylic acid methyl ester(18.8 mmol, 4.94 g) in a mixture of THF (38 mL) and methanol (38 mL) wasadded 1N sodium hydroxide (38 mL) and the mixture was heated to 50-55 °C. After 15 h, TLC analysis of the reaction mixture indicated theabsence of starting material and the mixture was allowed to cool to roomtemperature. The solvent was removed under vacuum and the residue wasdiluted with water (100 mL) and extracted with diethyl ether (2×50 mL)to remove any neutral impurities. Then, the basic aqueous layer wasacidified with 1N hydrochloric acid and the product was extracted withethyl acetate (2×75 mL). The combined extracts were washed with brine,dried (MgSO₄), filtered and the solution was concentrated in vacuo. Theresidual material was dried under high vacuum to afford 4.31 g (92%) of1-[4-(methylsulfonyl)butyl]cyclopentane carboxylic acid as a low meltingcolorless solid. LR MS (C11H20O4S): 249 (M+H).

Example 44 1-[4-(methylthio)butyl]cyclopentanecarboxylic acid.

[0305]

[0306] To a solution of 1-[4-(methylthio)butyl]cyclopentanecarboxylicacid methyl ester (18.8 mmol, 4.94 g) in a mixture of THF (38 mL) andmethanol (38 mL) was added 1N sodium hydroxide (38 mL) and the mixturewas heated to 50-55° C. for 15 h. TLC analysis of the reaction mixturedid not detect the presence of starting ester and the reaction wascooled to room temperature. The volatiles were removed under reducedpressure and the residue was diluted with water (100 mL) and wasextracted with diethyl ether (2×50 mL) to remove any neutral impurities.Then the separated aqueous layer was acidified with 1N hydrochloric acidand the mixture was extracted with ethyl acetate (2×75 mL). The combinedethyl acetate extracts were washed with brine, dried (MgSO₄), filtered,then concentrated in vacuo and the residue was dried under high vacuumto afford 4.31 g (92%) of 1-[4-(methylthio)butyl]cyclopentanecarboxylicacid as a low melting colorless solid. HR MS: Obs. mass, 216.1181.Calcd. mass, 216.1184, M+.

Example 45N-[[1-[4-(methylthio)butyl]cyclopentyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester.

[0307]

[0308] To a suspension of 4-nitro-L-phenylalanine methyl esterhydrochloride salt (181.84 mmol, 47.41 g),1-[4-(methylthio)butyl]cyclopentanecarboxylic acid (177.17 mmol, 38.33g) in DMF (470 mL) were added HBTU (177.17 mmol, 67.2 g) anddiisopropylethylamine (443 mmol, 77 mL) at room temperature. The clearsolution was stirred for 15 h at room temperature at which time TLCanalysis of the mixture indicated the absence of starting materials. Thereaction mixture was diluted with 600 mL of ethyl acetate then waswashed in turn with 0.5N hydrochloric acid (2×250 mL), saturated sodiumbicarbonate solution (2×250 mL) and brine (300 mL). The dried (MgSO₄)organic layer was filtered and evaporated to dryness under reducedpressure. The crude product which was purified by silica gel columnchromatography to afford 58.5 g (78%) ofN-[[1-[4-(methylthio)butyl]cyclopentyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester as an amorphous colorless solid. HRMS: Obs. mass, 423.1940.Calcd. mass, 423.1953 (M+H).

Example 464-nitro-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-L-phenylalaninemethyl ester.

[0309]

[0310] To a solution ofN-[[1-[4-(methylthio)butyl]cyclopentyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester (138.4 mmol, 58.5 g) in dichloromethane (1.2 L) was addedm-chloroperbenzoic acid (415 mmol, 71.7 g) at −5° C. (ice-salt bath).The suspension was stirred for 30 min at 0° C. and allowed to warm toroom temperature. After 5 h, analysis of the reaction by TLC indicatedthat the starting material was gone. The precipitated solid was removedby filtration and the filtrate was concentrated under vacuum to afford acolorless residue. The residue was dissolved in ethyl acetate (600 mL)and was washed with saturated sodium bicarbonate solution (3×300 mL).TLC analysis showed the presence of m-chloroperbenzoic acid.Accordingly, the ethyl acetate layer was washed in turn with saturatedsodium bisulfite solution (20 g in 150 mL of water), saturated sodiumbicarbonate solution (200 mL) and brine (300 mL). The dried (MgSO₄)ethyl acetate layer was filtered and evaporated to dryness to give acrude product which was dissolved in ethyl acetate. Diethyl ether andhexane were added to precipitate an oily residue. Some of the solventwas removed under reduced pressure to obtain a white suspension. Thesuspension was further diluted with diethyl ether and the resultingsolid was collected by filtration and was washed with hexane. Thecolorless low melting solid was dried to furnish 53.9 g (86%) ofN-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester, mp 40-44° C. HRMS: Obs. mass, 455.1854. Calcd. mass,455.1852 (M+H).

Example 47N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester.

[0311]

[0312] To a solution ofN-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester (33 mmol, 15 g) in toluene (100 mL, stored over 4 Åmolecular sieves) and freshly distilled THF (50 mL) was added Lawesson'sreagent (33 mmol, 13.35 g, 1.0 equiv.) at room temperature. The solutionwas heated to 60-65° C. and was stirred for 48 h at which time TLCanalysis of the mixture indicated the absence of starting material. Thereaction mixture was cooled to room temperature and was poured intosaturated sodium bicarbonate solution (200 mL) and extracted with ethylacetate (3×150 mL). An oil formed in the aqueous layer, which wasseparated, diluted with water and extracted with ethyl acetate (2×50mL). The combined ethyl acetate extracts were washed with saturatedsodium bicarbonate solution (200 mL), with brine (300 mL), dried(MgSO₄), filtered and the solution was concentrated in vacuo. Theresidual light brown syrup was purified by silica gel columnchromatography (hexane:ethyl acetate, 1:1) to obtain 6.87 g (44%) ofN-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester as a fluffy yellow solid. HRMS: Obs. mass, 493.1438. Calcd.mass, 493.1443 (M+Na).

Example 484-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl)-L-phenylalaninemethyl ester.

[0313]

[0314] The poorly solubleN-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester (19.3 mmol, 9.07 g) was dissolved in methanol (150 mL) andTHF (20 mL) by gentle heating with a heat gun. To this solution, zincdust (325 mesh, 193 mmol, 12.62 g, 10 equiv.) and ammonium chloride(289.5 mmol, 15.5 g, 15 equiv.) were added followed by water (75 mL) atroom temperature. After the addition of water, an exothermic reactionensued and the temperature rose to 45 to 50° C. The suspension wasstirred for 1 h, at which time TLC analysis of the mixture indicated theabsence of starting material. The reaction mixture was filtered and thefilter cake was washed with methanol (200 mL) and THF (100 mL). Thevolatiles were removed under vacuum and the organic residue wasextracted into ethyl acetate (2×200 mL). The combined extracts werewashed brine (250 mL) then were dried (MgSO₄), filtered and evaporatedto dryness to give 8.37 g (98%) of4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl)-L-phenylalaninemethyl ester as a colorless gum which was used directly for next step.HRMS: Obs. mass, 441.1884. Calcd. mass, 441.1882 (M+H).

Example 494-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester.

[0315]

[0316] To a solution of4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester (19.0 mmol, 8.37 g) and 2,6-dichlorobenzoyl chloride (21mmol, 4.4 g) in dichloromethane (90 mL) was added diisopropylethylamine(32.3 mmol, 5.6 mL) at room temperature. The solution was stirred for 15h at which time TLC analysis of the mixture indicated the absence ofstarting material. Then, it was diluted with water (100 mL) and the twolayers were separated. The aqueous phase was extracted withdichloromethane (100 mL) and the combined extracts were washed withbrine (200 mL). The dried (MgSO₄) solution was concentrated under vacuumand the residue was purified by silica gel column chromatography elutingwith (hexane:ethyl acetate:dichloromethane, 1:1:1) to obtain 11.54 g(99%) of4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester as a colorless solid, mp 200-202° C. HRMS: Obs. mass,613.1367. Calcd. mass, 613.1363 (M+H).

Example 504-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine.

[0317]

[0318] To a solution of4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester (25.86 mmol, 15.87 g) in ethanol (75 mL) was added aqueous1.0N sodium hydroxide (60 mL) at 50° C . The mixture was heated to50-55° C. and the resulting clear light brown solution was stirred for22 h at which time TLC analysis of the mixture indicated the absence ofstarting material. The mixture was diluted with water and allowed tocool to room temperature and was filtered to remove a small amount ofsolids. The filtrate was concentrated and the residual aqueous solutionwas washed with diethyl ether (2×75 mL). The basic aqueous layer wasacidified with 3.0N HCl to form a cloudy suspension and was extractedwith ethyl acetate (3×100 mL). The combined ethyl acetate extracts werewashed with brine (200 mL) then the dried (MgSO₄) solution was filteredand evaporated to dryness. The residue was taken up in dichloromethaneand diluted with diethyl ether:hexane (1:1) to obtain a solid which wascollected by filtration. Trituration of the solid with hot ethyl acetate(˜100 mL) resulted in a suspension that was then diluted with diethylether (˜50 mL) and the solid was collected by filtration. The aboveTrituration was repeated to afford 10.89 g (70%) of4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanineas a colorless solid. HR MS: Obs. mass, 599.1193. Calcd. mass, 599.1208(M+H).

Example 514-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalaninesodium salt.

[0319]

[0320] A suspension of4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]-cyclopentyl]thioxomethyl]-L-phenylalanine(16.49 mmol, 9.89 g) in water (100 mL) was treated with aqueous 1.0Nsodium hydroxide (16.4 mmol, 16.4 mL) at room temperature. The mixturewas heated to 40-45° C. and some acetonitrile (˜15 mL) was added to givean essentially clear solution containing a small amount of suspendedsolid. The solution was filtered and the filtrate was lyophilized toafford 10.1 g of sodium salt as a colorless solid. HRMS: Obs. mass,621.1023. Calcd. mass, 621.1027 (M+H).

Example 524-[[(2,4-dimethylpyridin-3-yl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester.

[0321]

[0322] To an ice cold solution of 2,4-dimethyl-3-pyridinecarboxylic acid(0.3 mmol, 45 mg) in dichloromethane (2 mL) containing one drop of DMF,was added oxalyl chloride (0.39 mmol, 49.5 mg) at 0° C. The reactionmixture was stirred for 30 min at this temperature, was allowed to warmto room temperature and was stirred for an additional 2 h. The solutionwas concentrated and the residue was dried under high vacuum. To amixture of above acid chloride and4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester (0.2 mmol, 88 mg) in dichloromethane (3 mL) was addeddiisopropylethylamine (1 mmol, 0.175 mL) at room temperature. After thesolution had stirred for 15 h, TLC analysis of the mixture did notdetect any starting material remaining. The solution was diluted withwater (20 mL) and dichloromethane (20 mL) and the layers were separated.The aqueous phase was extracted with dichloromethane (10 mL) and thecombined organic extracts were washed with brine (300 mL). The dried(MgSO₄) solution was filtered and evaporated to dryness and the residualmaterial was purified by RP-HPLC to obtain 74 mg (65%) of4-[[(2,4-dimethylpyridin-3-yl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester as an amorphous colorless solid. HRMS: Obs. mass, 574.2389.Calcd. mass, 574.2409 (M+H).

Example 534-[[(2,4-dimethylpyridin-3-yl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]-cyclopentyl]thioxomethyl]-L-phenylalaninetrifluoroacetic acid salt.

[0323]

[0324] To a solution of4-[[(2,4-dimethylpyridin-3-yl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalaninemethyl ester (0.118 mmol, 68 mg) in ethanol (4 mL) was added aqueous1.0N sodium hydroxide (3 mL) at room temperature and the stirred mixturewas heated to 45-50° C. After 3 h TLC analysis of the clear solutionindicated that the starting material had been consumed. The mixture wasconcentrated and the crude residue was purified by RP-HPLC to afford54.5 mg (82%) of4-[[(2,4-dimethylpyridin-3-yl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]-cyclopentyl]thioxomethyl]-L-phenylalaninetrifluoroacetic acid salt as an amorphous colorless solid. HR MS: Obs.mass, 560.2240. Calcd. mass, 560.2253 (M+H).

Example 54 1-(4-bromobutyl)cyclobutanecarboxylic acid ethyl ester.

[0325]

[0326] Using the general procedure described in example 40, startingwith cyclobutanecarboxylic acid ethyl ester,1-(4-bromobutyl)cyclobutanecarboxylic acid ethyl ester was prepared in58% yield as a colorless oil. HR MS: Obs. mass, 263.0563. Calcd. mass,263.0568, M+.

Example 55 1-[4-(methylthio)butyl]cyclobutanecarboxylic acid ethylester.

[0327]

[0328] Using the general procedure described in example 41, startingwith 1-(4-bromobutyl)cyclobutanecarboxylic acid ethyl ester,1-[4-(methylthio)butyl]cyclobutanecarboxylic acid ethyl ester wasprepared in 87% yield as a colorless oil. HR MS: Obs. mass, 230.1339.Calcd. mass, 230.1340, M+.

Example 56 ethyl 1-[4-(methylsulfonyl)butyl]cyclobutane carboxylic acidethyl ester.

[0329]

[0330] Using the general procedure described in example 46, startingwith 1-[4-(methylthio)butyl]cyclobutanecarboxylic acid ethyl ester,1-[4-(methylsulfonyl)butyl]cyclobutanecarboxylic acid ethyl ester wasprepared in 92% yield as a colorless oil. HR MS: Obs. mass, 262.1231.Calcd. mass, 262.1238, M+.

Example 57 1-[4-(methylsulfonyl)butyl]cyclobutanecarboxylic acid.

[0331]

[0332] Using the general procedure described in example 43, startingwith 1-[4-(methylsulfonyl)butyl]cyclobutanecarboxylic acid ethyl ester,1-[4-(methylsulfonyl)butyl] cyclobutanecarboxylic acid was prepared in92% yield as a low melting colorless solid. HR MS: Obs. mass, 234.0921.Calcd. mass, 234.0918, (M+).

Example 58N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester.

[0333]

[0334] Using the general procedure described in example 45, startingwith 1-[4-(methylsulfonyl)butyl]cyclobutanecarboxylic acid and4-nitro-L-phenylalanine methyl ester hydrochloride salt,N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester was prepared in 89% yield as a yellow gum. HR MS: Obs.mass, 441.1700. Calcd. mass, 441.1696 (M+H).

Example 59N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-4-nitro-L-phenylalaninemethyl ester.

[0335]

[0336] Using the general procedure described in example 47, startingwith4-nitro-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]carbonyl]-L-phenylalaninemethyl ester,N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-4-nitro-L-phenylalaninemethyl ester was prepared in 80% yield as a colorless solid, mp 150-152°C. HR MS: Obs. mass, 457.1464. Calcd. mass, 457.1467, (M+H).

Example 604-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-L-phenylalaninemethyl ester.

[0337]

[0338] Using the general procedure described in example 48, startingwithN-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-4-nitro-L-phenylalaninemethyl ester,4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-L-phenylalaninemethyl ester was prepared in 94% yield as a hygroscopic solid. HR MS:Obs. mass, 427.1720. Calcd. mass, 427.1725, (M+H).

Example 614-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester.

[0339]

[0340] Using the procedure described in example 49, starting with4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-L-phenylalaninemethyl ester,4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester was obtained in 92% yield as an amorphous colorless solid.HR MS: Obs. mass, 599.1207. Calcd. mass, 599.1208 (M+H).

Example 624-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.

[0341]

[0342] Using the general procedure described in example 50, startingwith4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester,4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalaninewas prepared in 99% yield as an amorphous colorless solid. HR MS: Obs.mass, 585.1038. Calcd. mass, 585.1051 (M+H).

Example 63N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-4-[[[4-(trifluoromethyl)pyrimidin-5-yl]carbonyl]amino]- L-phenylalanine methyl ester.

[0343]

[0344] Using the general procedure described in example 45, startingwith 4-(trifluoromethyl)pyrimidine-5-carboxylic acid and4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl)-L-phenylalaninemethyl ester, N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-4-[[[2-(trifluoromethyl) pyrimidin-5-yl]carbonyl]amino]-L-phenylalaninemethyl ester was prepared in 32% yield as an amorphous colorless solid.HR MS: Obs. mass, 601.1766. Calcd. mass, 601.1766 (M+H).

Example 64N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-4-[[[2-(trifluoromethyl)pyrimidin-5-yl]carbonyl]amino]-L-phenylalanine.

[0345]

[0346] Using the general procedure described in example 50, startingwithN-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-4-[[[2-(trifluoromethyl)pyrimidin-5-yl]carbonyl]amino]-L-phenylalanine methyl ester,N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-4-[[[2-(trifluoromethyl)pyrimidin-5-yl]carbonyl]amino]-L-phenylalanine was prepared in 22% yieldas an amorphous colorless solid. HR MS: Obs. mass, 587.1619. Calcd.mass, 587.1609 (M+H).

Example 65 Preparation 1-(3-bromopropyl)cyclobutanecarboxylic acid ethylester.

[0347]

[0348] Using the general procedure described in example 40, startingwith cyclobutanecarboxylic acid ethyl ester and 1,3-dibromopropane,1-(3-bromopropyl)cyclobutanecarboxylic acid ethyl ester was prepared in33% yield as a colorless oil. HR MS: Obs. mass, 248.0416. Calcd. mass,248.0412 (M+).

Example 66 1-[3-(methylthio)propyl]cyclobutanecarboxylic acid ethylester and 1-[3-(methylthio)propyl]cyclobutanecarboxylic acid.

[0349]

[0350] Using the general procedure described in example 41, startingwith 1-(3-bromopropyl)cyclobutanecarboxylic acid ethyl ester,1-[3-(methylthio)propyl]cyclobutanecarboxylic acid ethyl ester wasprepared in 58% yield as a colorless oil. HR MS: Obs. mass, 216.1182.Calcd. mass, 216.1184 (M+). Also,1-[3-(methylthio)propyl]cyclobutanecarboxylic acid was obtained in 16%yield as a colorless oil. HR MS: Obs. mass, 188.0872. Calcd. mass,188.0871 (M+).

Example 67N-[[1-[3-(methylthio)propyl]cyclobutyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester.

[0351]

[0352] Using the general procedure described in example 45, startingwith 4-nitro-L-phenylalanine methyl ester hydrochloride salt and1-[3-(methylthio)propyl]cyclobutanecarboxylic acid,N-[[1-[3-(methylthio)propyl]cyclobutyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester was prepared in 92% yield as an yellow viscous oil. HR MS:Obs. mass, 395.1638. Calcd. mass, 395.1640 (M+H).

Example 68N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester.

[0353]

[0354] Using the general procedure described in example 47, startingwith4-nitro-N-[[1-[3-(methylthio)propyl]cyclobutyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester,N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester was prepared in 95% yield as a colorless viscous oil. HRMS: Obs. mass, 411.1408. Calcd. mass, 411.1412 (M+H).

Example 694-amino-N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester.

[0355]

[0356] Using the general procedure described in example 48, startingwith N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-4-nitro-L-phenylalanine methyl ester,4-amino-N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester was prepared in 97% yield as an hygroscopic yellow solid.HR MS: Obs. mass, 381.1660. Calcd. mass, 381.1671 (M+H).

Example 70 4-[(2,6-dichlorophenylcarbonyl)amino]-N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylalanine methyl ester.

[0357]

[0358] Using the general procedure described in example 49, startingwith4-(amino)-N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester and 2,6-dichlorobenzoyl chloride,4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester was prepared in 83% yield as a colorless solid, mp 184-186°C. HR MS: Obs. mass, 553.1139. Calcd. mass, 553.1153 (M+H).

Example 714-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylthio)propyl]cyclobutyl]-thioxomethyl]-L-phenylalanine.

[0359]

[0360] Using the general procedure described in example 50, startingwith4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester,4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninewas prepared in 97% yield as a colorless solid, mp 186-188° C. HR MS:Obs. mass, 539.0986. Calcd. mass, 539.0996 (M+H).

Example 72 1-[3-(methylsulfonyl)propyl]cyclobutanecarboxylic acid ethylester.

[0361]

[0362] Using the general procedure described in example 46, startingwith 1-[3-(methylthio)propyl]cyclobutanecarboxylic acid ethyl ester,1-[3-(methylsulfonyl)propyl]cyclobutanecarboxylic acid ethyl ester wasprepared in 87% yield as a colorless oil. HR MS: Obs. mass, 248.1084.Calcd. mass, 248.1082 (M+).

Example 73 1-[3-(methylsulfonyl)propyl]cyclobutanecarboxylic acid.

[0363]

[0364] Using the general procedure described in example 43, startingwith 1-[3-(methylsulfonyl)propyl]cyclobutanecarboxylic acid ethyl ester,1-[3-(methylsulfonyl)propyl]cyclobutanecarboxylic acid was prepared in76% yield as a colorless solid, mp 113-116° C. HR MS: Obs. mass,220.0770. Calcd. mass, 220.0769 (M+).

Example 74N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester.

[0365]

[0366] Using the general procedure described in example 45, startingfrom 4-nitro-L-phenylalanine methyl ester and1-[3-(methylsulfonyl)propyl]cyclobutane carboxylic acid,N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]carbonyl]-4-nitro-L-phenylalanine methyl ester wasprepared in 76% yield as a colorless amorphous solid. HR MS: Obs. mass,427.1526. Calcd. mass, 427.1539 (M+H).

Example 75N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester.

[0367]

[0368] Using the general procedure described in example 47, startingwithN-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]carbonyl]-4-nitro-L-phenylalaninemethyl ester,N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester was prepared in 88% yield as an yellow sticky solid. HR MS:Obs. mass, 443.1309. Calcd. mass, 443.1310 (M+H).

Example 764-amino-N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester.

[0369]

[0370] Using the general procedure described in example 48, startingwithN-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-4-nitro-L-phenylalaninemethyl ester,4-amino-N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester was prepared in 97% yield as an hygroscopic yellow solid.HR MS: Obs. mass, 413.1556. Calcd. mass, 413.1570 (M+H).

Example 774-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester.

[0371]

[0372] Using the general procedure described in example 49, startingwith4-amino-N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester and 2,6-dichlorobenzoyl chloride,4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester was prepared in 82% yield as a colorless amorphous solid.HR MS: Obs. mass, 585.1056. Calcd. mass, 585.1051 (M+H).

Example 784-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalanine.

[0373]

[0374] Using the general procedure described in example 50, startingwith4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester,4-[(2,6-dichlorophenylcarbonyl)amino]-N-[[1-[3-(methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalaninewas prepared in 87% yield as an amorphous colorless solid. HR MS: Obs.mass, 571.0894. Calcd. mass, 571.0895 (M+H).

Example 79 2-chloro-5-(trifluoromethyl)phenol triflate.

[0375]

[0376] To a solution of 2-chloro-5-(trifluoromethyl)phenol (24.4 mmol,4.8 g) in dichloromethane (160 mL) was added DMAP (54.0 mmol, 6.7 g) at−70 ° C. followed by triflic anhydride (36.6 mmol, 10.32 g, 6.16 mL) at−70° C. After the addition was complete, the suspension was stirred for30 min at this temperature and then warmed to room temperature. Afteranother 3 h, when starting material could not be detected by TLC of thereaction mixture indicated the absence of starting material, the stirredmixture was diluted with H₂O (100 mL) and the two layers were separated.

[0377] The aqueous layer was extracted with dichloromethane (100 mL)andthen the combined dichloromethane extracts were washed with brine. Thedried (MgSO₄) solution was filtered and evaporated to dryness to give acolorless residue which was purified by silica gel column chromatography(hexane:diethyl ether 4:1) to obtain 6.8 g (85%) of2-chloro-5-(trifluoromethyl)phenol triflate as a colorless oil. HR MS:Obs. mass, 327.9388. Calcd. mass, 327.9392 (M+).

Example 80 2-chloro-5-(trifluoromethyl)benzoic acid.

[0378]

[0379] A 250 mL pressure bottle was charged with2-chloro-5-(trifluoromethyl)phenol triflate (20.6 mmol, 6.76 g),Pd(OAc)₂ (1.71 mmol, 384 mg) and dppp (1.71 mmol, 701 mg). The flask wasclosed with a septum and evacuated three times with argon. Acetonitrile(114 mL), triethylamine (225.3 mmol, 30.7 mL) and water (22.2 mL) wereadded in succession with the aid of a syringe. The rubber septum wasreplaced with a teflon lined lid. The flask was pressurized with carbonmonoxide (40 psi) and the gas was released. This process was repeatedthree times and finally the reaction was stirred for 5 min underpressure. The flask was then disconnected from the gas cylinder,immersed in a preheated oil bath (83-85° C.) and stirred for 2 h. Theflask was re-pressurized with carbon monoxide and stirred for another 1h. After the reaction mixture was cooled to room temperature, thepressure was released and it diluted with diethyl ether (250 mL) and 25mL of 1.0N NaOH. The sodium salt was extracted into water (2×100 mL).The combined aqueous extracts were acidified with 1.0N HCl and extractedwith diethyl ether (3×100 mL). The combined diethyl ether extracts werewashed with brine, dried (MgSO₄), filtered and the solution wasconcentrated in vacuo to furnish a light yellow solid. The solid wasdissolved in diethyl ether (100 mL) and extracted with 1.0N NaOHsolution (2×50 mL). The combined aqueous layers were acidified andextracted with diethyl ether (2×100 mL). After the combined organicextracts were washed with brine (100 mL), the dried (MgSO₄) solution wasfiltered and evaporated to dryness to give 1.6 g (35%) of2-chloro-5-(trifluoromethyl)benzoic acid obtained as a colorless solid,mp 82-83.5° C. HR MS: Obs. mass, 223.9852. Calcd. mass, 223.9851 (M+).

Example 814-[[[(2-chloro-5-(trifluoromethyl)phenyl]carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester.

[0380]

[0381] Using the general procedure described in example 52, startingwith 4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine methyl ester and2-chloro-5-(trifluoromethyl)benzoic acid,4-[[[(2-chloro-5-(trifluoromethyl)phenyl]carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester was prepared in 97% yield as a colorless amorphous solid.HR MS: Obs. mass, 633.1477. Calcd. mass, 633.1471 (M+H).

Example 824-[[[(2-chloro-5-(trifluoromethyl)phenyl]carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.

[0382]

[0383] Using the general procedure described in example 50, startingwith 4-[[[(2-chloro-5-(trifluoromethyl)phenyl]carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]-cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester,4-[[[(2-chloro-5-(trifluoromethyl)phenyl]carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine was prepared in 75% yieldas an amorphous colorless solid. HR MS: Obs. mass, 619.1315. Calcd.mass, 619.1318 (M+H).

Example 834-[[[(2,6-dimethyl-4-(trifluoromethyl)pyridin-3-yl4carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester.

[0384]

[0385] To suspension of2,6-dimethyl-4-(trifluoromethyl)pyridine-3-carboxylic acid (0.84 mmol,184 mg) in dichloromethane (10 mL) containing DMF (3 drops) was addeddropwise oxalyl chloride (1.14 mmol, 146 mg, 0.1 mL) at 0° C. for 2-3min. After the addition was complete, the reaction was stirred for 30min at 0° C. and then allowed to warm to room temperature. The clearsolution was stirred for another 2 h at room temperature, then thesolvent was removed under reduced pressure and the residue was driedunder high vacuum for 1 h. To a mixture of4-amino-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester (0.7 mmol, 298 mg) and amberlyst A-21 (1.4 mmol, 900 mg) inethyl acetate (10 mL, stored over 4Å molecular sieves) in a 4-neckedsonicator flask was added a solution of the above prepared acid chloridein ethyl acetate (6 mL) at room temperature. The mixture was subjectedfor sonication for 30 min and then was partitioned between water (100mL) and ethyl acetate (100 mL). The separated aqueous layer wasextracted with ethyl acetate (50 mL) and the combined extracts werewashed brine (100 mL. The dried (MgSO₄) ethyl acetate layer was filteredand evaporated to dryness in vacuo and the residue was purified byRP-HPLC to obtain 139 mg (32%) of4-[[[(2,6-dimethyl-4-(trifluoromethyl)pyridin-3-yl]carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester as an amorphous colorless solid. HR MS: Obs. mass,628.2122. Calcd. mass, 628.2127 (M+H).

Example 844-[[[(2,6-dimethyl-4-(trifluoromethyl)pyridin-3-yl]carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.

[0386]

[0387] To a suspension of4-[[[(2,6-dimethyl-4-(trifluoromethyl)pyridin-3-yl]carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester (0.2 mmol, 125 mg) in ethanol (7 mL) was added a 1.0Nsolution of sodium hydroxide (5.0 mL) at room temperature. Within fewminutes the reaction mixture become a clear solution and it was heatedto 45-50° C. and stirred for 4 hr, at which time TLC analysis of themixture indicated the absence of starting material. After the solutionwas cooled to room temperature, the ethanol was removed in vacuo and theresidue was purified by RP-HPLC to obtain 67.5 mg (55%) of4-[[[(2,6-dimethyl-4-(trifluoromethyl)pyridin-3-yl]carbonyl]amino]-N-[[1-[4-(methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalaninemethyl ester as an amorphous colorless solid. HR MS: Obs. mass,614.1970. Calcd. mass, 614.1970 (M+H).

Example 854-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-bromophenyl)thioxomethyl]-L-phenylalanine.

[0388]

[0389] Using the method described in examples 35 to 39, and startingwith 2-bromobenzoic acid and 4-nitro-L-phenylalanine methyl esterhydrochloride salt, the title compound was prepared. HRMS Obs. mass,550.9593. Calcd mass, 550.9598 (M+H).

Example 864-[(2S,4R)-3-acetyl-5-oxo-2-phenyl-4-(phenylmethyl)imidazolidin-1-yl]-N-[[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanineand4-[(2R,4R)-3-acetyl-5-oxo-2-phenyl-4-(phenylmethyl)imidazolidin-1-yl]-N-[[4-(methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine.

[0390]

[0391] Using the procedure described in examples 26 to 29, the titlecompounds were prepared. The isomers were separated by chromatography atthe methyl ester stage. For the 2S,4R isomer, HRMS, Obs. mass, 650.2670.Calcd mass, 650.2665 (M+Na). For the 2R,4R isomer, HRMS, obs. mass,650.2679. Calcd. mass, 650.2665 (M+Na).

[0392] Assays:

[0393] 1. VLA-4/VCAM-1 Screening Assay

[0394] VLA-4 antagonist activity, defined as ability to compete forbinding to immobilized VCAM-1, was quantitated using a solid-phase, dualantibody ELISA. VLA-4 (α4β1 integrin) bound to VCAM-1 is detected by acomplex of anti-integrin β1 antibody: HRP-conjugated anti-mouse IgG:chromogenic substrate (K-Blue). Initially, this entailed coating 96 wellplates (Nunc Maxisorp) with recombinant human VCAM-1 (0.4 μg in 100 μlPBS), sealing each plate and then allowing the plates to stand at 4° C.for ¹⁸18 hr. The VCAM-coated plates were subsequently blocked with 250μl of 1% BSA/0.02% NaN₃ to reduce non-specific binding. On the day ofassay, all plates are washed twice with VCAM Assay Buffer (200 μl/wellof 50 mM Tris-HCl, 100 mM NaCl, 1 mM MnCl₂, 0.05% Tween 20; pH 7.4).Test compounds are dissolved in 100% DMSO and then diluted 1:20 in VCAMAssay Buffer supplemented with 1 mg/mL BSA (i.e., final DMSO=5%). Aseries of 1:4 dilutions are performed to achieve a concentration rangeof 0.005 nM - 1.563 μM for each test compound. 100 μl per well of eachdilution is added to the VCAM-coated plates, followed by 10 μl of Ramoscell-derived VLA-4. These plates are sequentially mixed on a platformshaker for 1 min, incubated for 2 hr at 37° C., and then washed fourtimes with 200 μl/well VCAM Assay Buffer. 100 μl of mouse anti-humanintegrin β1 antibody is added to each well (0.6 μg/mL in VCAM AssayBuffer +1 mg/mL BSA) and allowed to incubate for 1 hr at 37° C. At theconclusion of this incubation period, all plates are washed four timeswith VCAM Assay Buffer (200 μl/well). A corresponding second antibody,HRP-conjugated goat anti-mouse IgG (100 μl per well @ 1:800 dilution inVCAM Assay Buffer +1 mg/mL BSA), is then added to each well, followed bya 1 hr incubation at room temperature and concluded by three washes (200μl/well) with VCAM Assay Buffer. Color development is initiated byaddition of 100 μl K-Blue per well (15 min incubation, room temp) andterminated by addition of 100 μl Red Stop Buffer per well. All platesare then read in a UV/Vis spectrophotometer at 650 nM. Results arecalculated as % inhibition of total binding (i.e., VLA-4+VCAM-1 in theabsence of test compound). Selected data for compounds of this inventionare shown in the table below:

[0395] 2. Ramos (VLA-4)/VCAM-1 Cell-Based Screening Assay Protocol

[0396] Materials:

[0397] Soluble recombinant human VCAM-1 (mixture of 5- and 7-Ig domain)was purified from CHO cell culture media by immunoaffinitychromatography and maintained in a solution containing 0.1M Tris-glycine(pH 7.5), 0.1M NaCl, 5 mM EDTA, 1 mM PMSF, 0.02% 0.02% NaN₃ and 10 μg/mLleupeptin. Calcein-AM was purchased from Molecular Probes Inc.

[0398] Methods:

[0399] VLA-4 (α4β1 integrin) antagonist activity, defined as ability tocompete with cell-surface VLA-4 for binding to immobilized VCAM-1, wasquantitated using a Ramos-VCAM-1 cell adhesion assay. Ramos cellsbearing cell-surface VLA-4, were labeled with a fluorescent dye(Calcein-AM) and allowed to bind VCAM-1 in the presence or absence oftest compounds. A reduction in fluorescence intensity associated withadherent cells (% inhibition) reflected competitive inhibition of VLA-4mediated cell adhesion by the test compound.

[0400] Initially, this entailed coating 96 well plates (Nunc Maxisorp)with recombinant human VCAM-1 (100 ng in 100 μl PBS), sealing each plateand allowing the plates to stand at 4° C. for ^(˜)18 hr. The VCAM-coatedplates were subsequently washed twice with 0.05% Tween-20 in PBS, andthen blocked for 1 hr (room temperature) with 200 μl of Blocking Buffer(1% BSA/0.02% thimerosal) to reduce non-specific binding. Following theincubation with Blocking Buffer, plates were inverted, blotted and theremaining buffer aspirated. Each plate was then washed with 300 μl PBS,inverted and the remaining PBS aspirated.

[0401] Test compounds were dissolved in 100% DMSO and then diluted 1:25in VCAM Cell Adhesion Assay Buffer (4 mM CaCl₂, 4 mM MgCl₂ in 50 mMTRIS-HCl, pH 7.5) (final DMSO=4%). A series of eight 1:4 dilutions wereperformed for each compound (general concentration range of 1 nM -12,500 nM). 100 μl/well of each dilution was added to the VCAM-coatedplates, followed by 100 μl of Ramos cells (200,000 cells/well in 1%BSA/PBS). Plates containing test compounds and Ramos cells were allowedto incubate for 45 min at room temperature, after which 165 μl/well PBSwas added. Plates were inverted to remove non-adherent cells, blottedand 300 μl/well PBS added. Plates were again inverted, blotted and theremaining buffer gently aspirated. 100 μl Lysis Buffer (0.1% SDS in 50mM TRIS-HCl, pH 8.5) was added to each well and agitated for 2 min on arotary shaking platform. The plates were then read for fluorescenceintensity on a Cytofluor 2300 (Millipore) fluorescence measurementsystem (excitation=485 nm, emission=530 nm). The results are shown inthe following table:

[0402] 3. MadCAM RPMI 8866 Cell Based Assay

[0403] MadCAM binding activity was quantified using an RPMI 8866cell-based assay. RPMI 8866 cells bearing cell surface MadCAM werelabelled with fluorescent dye (calcein AM) and allowed to bind MadCAM inthe presence or absence of test compounds. A reduction in fluoresenceintensity associated with adherent cells (% inhibition) reflectscompetitive inhibition of MadCAM mediated cell adhesion by the testcompound.

[0404] Initially, this entailed coating 96 well plates (Nunc F96Maxisorp) with 25 ng/well of MadCAM (100 μl/well in coating buffer: 10mM carbonate/bicarbonate buffer, 0.8 g/L sodium carbonate, 1.55 g/Lsodium bicarbonate, adjusted to pH 9.6 with 1N HCL), sealing andwrapping each plate and refrigerating the plates for at least 24 hrs.The MadCAM-coated plates were subsequently washed twice with 0.05%Tween-20 in PBS, and then blocked for at least 1 hr. at room temperaturewith of blocking buffer (1% nonfat dry milk in PBS) to reducenon-specific binding. Following the incubation with blocking buffer,plates were washed with PBS, hand blotted, and the remaining liquidaspirated.

[0405] RPMI 8866 cells (2×10⁶ cells/ml×10 ml. per plate x number ofplates) were transferred to a 50 ml centrifuge tube filled with PBS andspun at 200 x g for 8 minutes, after which the PBS is poured off and thepellet resuspended to 10×10⁶ cells/ml in PBS. Calcein (diluted with 200μl DMSO from a 5 mg/ml frozen stock) was added to the cells at 5 μl/mlPBS. After incubation at 37 degrees C for 30 min. in the dark, the cellswere washed in PBS and resuspended at 2×106 cells/ml in cell buffer(RPMI 1640 medium (no additives)).

[0406] Test compounds were dissolved in 100% DMSO and then diluted 1:25in binding buffer (1.5 mM CaCl₂, 0.5 mM MnCl₂ in 50 mM TRIS-HCl,adjusted to pH 7.5 with NaOH). Remaining dilutions were into dilutionbuffer (4% DMSO in binding buffer—2% DMSO final when diluted 1:2 inwells). A series of dilutions were performed for each compound tested.129 μl of binding buffer was placed in the first row of wells in theMadCAM-coated plates. 100 μl/well of dilution buffer was added to theremaining wells, followed by 5.4 μl of each test compound in theappropriate dilution (in triplicate). 100 μl of cells (200,000cells/well) were added. Control wells contained 100 μl dilutionbuffer+100 μl cell buffer, and 100 μl dilution buffer+100 μl cellbuffer. Plates were allowed to incubate for 45 min at room temperature,after which 150 μl/well PBS was added. Plates were inverted to removenon-adherent cells, blotted and 200 μl/well PBS added. Plates were againinverted, blotted and the remaining buffer gently aspirated. 100 μl PBSwas added to each well. The plates were then read for fluorescenceintensity on a fluorescence measurement system (excitation=485 nm,emission=530 nm, sensitivity=2). A linear regression analysis wasperformed to obtain the IC₅₀ of each compound. The results are shown inthe following table: ELISA IC₅₀ nM Ramos Example (VLA/VCAM) IC₅₀ RPMIIC₅₀  3 4.0 66.5 10 1.5 33 11 5.6 42.5 18 0.47 60 25 6.0 101 29 220 344,180 39 784 50 30 53 148 62 8 8.7 64 87 71 926 78 341 82 84 5.5 83

Example 4 Acute airway inflammation in the atopic primate.

[0407] Airway inflammation in the monkey was determined using amodification of the protocol described by Turner et al. (Turner et al.,1994). Adult male cynomolgus monkeys (Macaca fascicularis, HazeltonLabs, Denver, Pa.) weighing between 3.6-5.8 kg were used in thesestudies. All animals exhibited positive skin and airway responses toAscaris suum antigen and had at least a 3-fold increase in thesensitivity to methacholine (MCh) when subjected to an aerosol ofascaris extract.

[0408] On the day of each experiment the animals were anesthetized withketamine hydrochloride, 12 mg/kg, and xylazine, 0.5 mg/kg, intubatedwith a cuffed endotracheal tube (3 mm, Mallinckrodt Medical, St. Louis,Mo.), then seated in an upright position in a specially designedPlexiglass chair (Plas-Labs, Lansing, Mich.). The endotracheal tube wasconnected to a heated Fleisch pneumotachograph. Airflow was measured viaa Validyn differential pressure transducer (DP 45-24) that was attachedto the pneumotachograph. Transpulmonary pressure was measured via asecond Validyne transducer (DP 45-24) connected between a sidearm of thetracheal cannula and a 18-gauge intrapleural needle inserted in theintercostal space located below the left nipple. Recordings of pressureand flow and the calculation of R_(L) were made using the ModularInstruments data acquisition system as described above. Baseline R_(L)was measured for all animals on the day of each experiment and had anaverage value of about 0.04 cmH₂O/ml/sec.

[0409] Protocol

[0410] Airway inflammation was induced by exposing the animal to anaerosol of A. Suum extract for 60 sec. The aerosol was delivered via anebulizer (De Vilbiss Model 5000, Healt Care Inc., Somerset, Pa.) thatwas attached to the endotracheal tube. The concentration of extract waspredetermined for each animal (500 to 50,000 PNU) and caused at least adoubling in the airway resistance. At 24 hour after the antigenchallenge, the animals were anesthetized as described previously andplaced on a stainless steel table. Airway inflammation was assessed byinserting a pediatric bronchoscope into the airway lumen down to aboutthe 4 or 5^(th) generation bronchi and gently lavaging with 3×2 mlaliquots of sterile Hanks Balanced Salt Solution. The recovered lavagefluid then was analyzed for the total cell and differential cell countsusing standard hematological techniques.

[0411] Drug Treatment

[0412] The animals received drug or a vehicle, p.o., administered 2hours prior to antigen challenge. The compound of example 1 caused asignificant decrease in the number and percent of inflammatory cellspresent in the lavage fluid relative to vehicle treated control animals.

1. A compound of the formula:

wherein X is a group of the formula

wherein: R₁₅ is halogen, nitro, lower alkyl sulfonyl, cyano, loweralkyl, lower alkoxy, lower alkoxycarbonyl, carboxy, lower alkylaminosulfonyl, perfluorolower alkyl, lower alkylthio, hydroxy loweralkyl, alkoxy lower alkyl, lower alkylthio lower alkyl, loweralkylsulfinyl lower alkyl, lower alkylsulfonyl lower alkyl, loweralkylsulfinyl, lower alkanoyl, aroyl, aryl, aryloxy; R₁₆ is hydrogen,halogen, nitro, cyano, lower alkyl, OH, perfluorolower alkyl, or loweralkylthio; or X is a group of formula X-2

wherein Het is a 5- or 6-membered heteroaromatic ring containing 1, 2 or3 heteroatoms selected from N,O, and S, or Het is a 9- or 10-memberedbicyclic heteroaromatic ring containing 1, 2, 3 or 4 heteroatomsselected from O, S, and N; R₁₅ and R₁₆ are as above; R₃₀ is hydrogen orlower alkyl; and p is an integer from 0 to 1 or X is a group of formulaX-3

wherein: R₁₈ is aryl, heteroaryl, aryl lower alkyl, heteroaryl loweralkyl R₁₉ is substituted or unsubstituted lower alkyl, aryl, heteroaryl,arylalkyl, heteroaryl alkyl, and R₂₀ is substituted or unsubstitutedlower alkanoyl or aroyl; and Y is a group of formula Y-1

wherein: R₂₂ and R₂₃ are independently hydrogen, lower alkyl, loweralkoxy, cycloalkyl, aryl, arylalkyl, nitro, cyano, lower alkylthio,lower alkylsulfinyl, lower alkyl sulfonyl, lower alkanoyl, halogen, orperfluorolower alkyl and at least one of R₂₂ and R₂₃ is other thanhydrogen, and R₂₄ is hydrogen, lower alkyl, lower alkoxy, aryl, nitro,cyano, lower alkyl sulfonyl, or halogen; or Y-2 is a group of theformula:

Het is a five or six membered heteroaromatic ring bonded via a carbonatom wherein said ring contains one, two or three heteroatoms selectedfrom the group consisting of N, O and S and R₃₀ and R₃₁ areindependently hydrogen, lower alkyl, cycloalkyl, halogen, cyano,perfluoroalkyl, or aryl and at least one of R₃₀ and R₃₁ is adjacent tothe point of attachment; p is an integer of from 0 to 1; or Y is a groupof formula Y-3

wherein: R₂₅ is lower alkyl, unsubstituted or fluorine substituted loweralkenyl, or a group of formula R₂₆—(CH₂)_(e)—, R₂₆ is aryl, heteroaryl,azido, cyano, hydroxy, lower alkoxy, lower alkoxycarbonyl, loweralkanoyl, lower alkylthio , lower alkyl sulfonyl, lower alkyl sulfinyl,perfluoro lower alkanoyl, nitro, or R₂₆ is a group of formula —NR₂₈R₂₉,wherein R₂₈ is H or lower alkyl, R₂₉ is hydrogen, lower alkyl, loweralkoxycarbonyl, lower alkanoyl, aroyl, perfluoro lower alkanoylamino,lower alkyl sulfonyl, lower alkylaminocarbonyl, arylaminocarbonyl, orR₂₈ and R₂₉ taken together form a 4, 5 or 6-membered saturatedcarbacyclic ring optionally containing one heteroatom selected from O,S, and N; with the carbon atoms in the ring being unsubstituted orsubstituted by lower alkyl or halogen, Q is —(CH₂)_(f)O—, —(CH₂)_(f)S—,—(CH₂)_(f)N(R₂₇)—, or —(CH₂)_(f), R₂₇ is H, lower alkyl, aryl, loweralkanoyl, aroyl or lower alkoxycarbonyl, e is an integer from 0 to 4,and f is an integer from 0 to 3; and the dotted bond is optionallyhydrogenated; and pharmaceutically acceptable salts and esters thereof.2. A compound of claim 1 wherein X is a group of the formula

and Y, R₁₅ and R₁₆ are as in claim
 1. 3. A compound of claim 2 whereinR₁₅ is lower alkyl, nitro, halogen, perfluoromethyl, or cyano and R₁₆ ishydrogen, lower alkyl, nitro, halogen, perfluoromethyl, or cyano.
 4. Acompound of claim 3 wherein R₁₅ and R₁₆ are independently chloro orfluoro.
 5. A compound of claim 3 wherein X-1 is selected from the groupof


6. A compound of claim 1 wherein X is a group of the formula X-2

and p, Y, R₁₅, R₁₆ and R₃₀ are as in claim
 1. 7. A compound of claim 6wherein Het is a 5- or 6-membered monocyclic heteroaromatic ringcontaining 1, 2 or 3 nitrogens, or a nitrogen and a sulfur, or anitrogen and an oxygen.
 8. A compound of claim 6 wherein Het is abicyclic heteroaromatic ring containing from 1 to 3 nitrogens.
 9. Acompound of claim 6 wherein R15 is nitro, lower alkyl sulfonyl, cyano,lower alkyl, lower alkoxy, perfluorolower alkyl, lower alkylthio, loweralkanoyl, or aryl.
 10. A compound of claim 9 wherein R₁₅ isunsubstituted phenyl.
 11. A compound of claim 6 wherein R₁₆ is hydrogen,halogen, nitro, cyano, lower alkyl, perfluoro lower alkyl; and R₃₀ ishydrogen or lower alkyl.
 12. A compound of claim 6 wherein Het is a 6membered monocyclic heteroaromatic ring containing 1 or 2 nitrogens or a10 membered bicyclic heteroaromatic ring containing one nitrogen, R₁₅ islower alkyl, or perfluoroalkyl and R₁₆ is hydrogen, lower alkyl, orperfluoroalkyl, and R₃₀ is absent.
 13. A compound of claim 6 wherein X-2is selected from the group of


14. A compound of claim 1 wherein X is a group of formula X-3

and Y, R₁₈, R₁₉, and R₂₀ are as in claim
 1. 15. A compound of claim 14wherein R₁₈ is phenyl.
 16. A compound of claim 14 wherein R₁₉ is loweralkyl which is unsubstituted or substituted by pyridyl or phenyl.
 17. Acompound of claim 14 wherein R₂₀ is substituted or unsubsituted loweralkanoyl.
 18. A compound of claim 14 wherein R₁₈ is phenyl, R₁₉ is loweralkyl which is unsubstituted or substituted by pyridyl or phenyl and R₂₀is lower alkanoyl.
 19. A compound of claim 14 wherein R₁₈ is phenylwhich is unsubstituted or substituted by halogen or lower alkoxy; R₁₉ isphenyl lower alkyl which is unsubstituted or substituted by loweralkoxy, pyridyl lower alkyl, or lower alkyl; and R₂₀ is substituted orunsubstituted lower alkanoyl.
 20. A compound of claim 19 wherein X-3 isselected from the group of


21. A compound of claim 1 wherein Y is a group of formula

and X, R₂₂, R₂₃, and R₂₄ are as in claim
 1. 22. A compound of claim 21wherein R₂₂ and R₂₃ are lower alkyl, trifluoromethyl, or halogen and R₂₄is hydrogen, lower alkyl, lower alkoxy, or halogen.
 23. A compound ofclaim 22 wherein Y-1 is selected from the group of


24. A compound of claim 1 wherein Y is a group of the formula Y-2

and p, X, Het, R₃₀ and R₃₁, are as in claim
 1. 25. A compound of claim24 wherein Het is a 6 membered heteroaromatic ring.
 26. A compound ofclaim 25 wherein the heteroatom is N.
 27. A compound of claim 26 whereinY-2 is selected from the group of


28. A compound of claim 1 wherein Y is a group of formula Y-3

and Y, R₂₅ and Q are as in claim 1, and the dotted bond can beoptionally hydrogenated.
 29. A compound of claim 28 wherein Y-3 isselected from the group of


30. A compound of claim 1 wherein X is a group of the formula X-1

and Y is a group of the formula Y-1


31. A compound of claim 1 wherein X is a group of the formula X-1

and Y is a group of the formula Y-2.
 32. A compound of claim 1 wherein Xis a group of the formula X-1

and Y is a group of the formula Y-3

wherein R₁₅, R₁₆, R₂₅ and Q are as above; and the dotted bond can beoptionally hydrogentated.
 33. A compound of claim 1 wherein X is a groupof the formula X-2

and Y is a group of the formula Y-1


34. A compound of claim 1 wherein X is a group of the formula X-2

and Y is a group of the formula Y-2.
 35. A compound of claim 1 wherein Xis a group of the formula X-2

and Y is a group of the formula Y-3

wherein R₁₅, R₁₆, R₂₅, R₃₀, Q and p are as above and the dotted bond canbe optionally hydrogenated.
 36. A compound of claim 1 wherein X is agroup of the formula X-3

and Y is a group of the formula Y-1


37. A compound of claim 1 wherein X is a group of the formula X-3

and Y is a group of the formula Y-2


38. A compound of claim 1 wherein X is a group of the formula X-3

and Y is a group of the formula Y-3

wherein R₁₈, R₁₉, R₂₀, R₂₅, and Q are as above and the dotted bond canbe optionally hydrogenated.
 39. A compound of claim 1 wherein X is agroup of the formula X-1

wherein R₁₆ is in the ortho position and is hydrogen, lower alkyl,nitro, cyano, halogen, lower alkylthio, perfluoroloweralkyl and R₁₅ islower alkyl, nitro, cyano, halogen, lower alkylsulfonyl,perfluoroloweralkyl, and Y is a group of the formula Y-1

where R₂₂ is hydrogen, halogen, trifluoroalkyl, or lower alkyl and R₂₃is halogen, trifluoroalkyl, or lower alkyl, and R24 is hydrogen or Y isa group of the formula Y-3

wherein Q is as above and the dotted bond can be optionallyhydrogenated; R₂₅ is R₂₆—(CH₂)e—; e is 2-4 and R₂₆ is azido, cyano,hydroxy, lower alkoxy, lower alkoxycarbonyl, lower alkanoyl, lower alkylsulfonyl, lower alkyl sulfinyl, perfluoro lower alkanoyl, nitro, orlower alkylthio or R₂₅ is NHR₂₉ where R₂₉ is lower alkanoyl or loweralkylamino carbonyl.
 40. A compound of claim 39 wherein X is a group ofthe formula X-1

wherein R₁₆ is in the ortho position and is hydrogen, lower alkyl,nitro, cyano, halogen, lower alkylthio, perfluoroloweralkyl and R₁₅ islower alkyl, nitro, cyano, halogen, lower alkylsulfonyl,perfluoroloweralkyl; and Y is a group of the formula Y-1

where R₂₂ is hydrogen, halogen, or lower alkyl and R₂₃ is halogen orlower alkyl, and R24 is hydrogen.
 41. A compound of claim 40 wherein R₁₆is hydrogen or halogen and R₁₅ is halogen; R22 is hydrogen, halogen,ethyl, or methyl and R₂₃ is halogen, ethyl, or methyl.
 42. A compound ofclaim 41 wherein R₁₆ is in the ortho position and R₁₅ and R16 are bothchlorine, and R₂₂ is methyl and R₂₃ is chlorine or ethyl.
 43. A compoundof claim 42 which is4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-chloro-6-methylphenyl)thioxomethyl]-L-phenylalanine.44. A compound of claim 39 wherein X is a group of the formula X-1

wherein R₁₆ is in the ortho position and is hydrogen, lower alkyl,nitro, cyano, halogen, lower alkylthio, perfluoroloweralkyl and R₁₅ islower alkyl, nitro, cyano, halogen, lower alkylsulfonyl,perfluoroloweralkyl; and Y is a group of the formula Y-3

which is a four to six membered cycloalkyl ring, R₂₅ is R₂₆—(CH₂)e—; eis 2-4 and R₂₆ is azido, cyano, hydroxy, lower alkoxy, loweralkoxycarbonyl, lower alkanoyl, lower alkyl sulfonyl, lower alkylsulfinyl, perfluoro lower alkanoyl, nitro, or lower alkylthio; and thedotted bond is hydrogenated.
 45. A compound of claim 44 wherein R₁₆ ishydrogen or halogen and R₁₅ is halogen; and Y-3 is a four or fivemembered ring and R₂₆ is lower alkoxy, lower alkyl sulfonyl, lower alkylsulfinyl, or lower alkylthio.
 46. A compound of claim 45 wherein R16 isin the ortho position and R₁₅ and R₁₆ are both chlorine, and R₂₆ islower alkyl sulfonyl or lower alkylthio.
 47. A compound of claim 46which is4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[[1-[(4-methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine.48. A compound of claim 39 wherein X is a group of the formula X-1

where R₁₆ is hydrogen or halogen and R₁₅ is halogen and Y is a group ofthe formula Y-1

where R₂₂ is hydrogen, halogen, ethyl, or methyl and R₂₃ is halogen,ethyl, or methyl and R₂₄ is hydrogen or Y is a group of the formula Y-3

where Y-3 is a four or five membered ring, R₂₅ is as in claim 39 and R₂₆is lower alkoxy, lower alkyl sulfonyl, lower alkyl sulfinyl, or loweralkylthio, and the dotted bond is optionally hydrogenated.
 49. Acompound of claim 48 wherein R₁₆ is in the ortho position and R₁₅ andR₁₆ are both chlorine, and when Y is Y-1 then R₂₂ is methyl and R₂₃ ischlorine or ethyl and when Y is Y-3, Y-3 is a four or five membered ringand R₂₆ is lower alkyl sulfonyl or lower alkylthio.
 50. A compound ofclaim 1 wherein Y is as in formula 1 and X is X-1

where R₁₅ is ortho and is halogen, lower alkyl, or perfluoroalkyl andR₁₆ is hydrogen, halogen, lower alkyl, or perfluoroalkyl.
 51. A compoundof claim 50 wherein R₁₅ is chlorine and R₁₆ is hydrogen or chlorine. 52.A compound of claim 1 wherein Y is as in formula 1 and X is X-2

where Het is pyridine or pyrimidine and R₁₅ is lower alkyl orperfluoroalkyl R₁₆, and R₂₀ are hydrogen, lower alkyl, orperfluoroalkyl.
 53. A compound of claim 1 wherein Y is as in formula 1and X is X-3

where R₁₉ is pyridinyl lower alkyl or phenyl lower alkyl, R₂₀ is loweralkanoyl, and R18 is phenyl.
 54. A compound of claim 1 wherein X is asin formula 1 and Y is Y-1

where R₂₂ is hydrogen or lower alkyl, R₂₃ is halogen, lower alkyl, orperfluoroalkyl, and R24 is hydrogen.
 55. A compound of claim 54 whereinR₂₂ is hydrogen or methyl and R₂₃ is halogen, ethyl, or trifluoromethyl.56. A compound of claim 1 wherein X is as in formula 1 and Y is Y-3 R₂₅

which is a four to six membered cycloalkyl ring, R₂₅ is R₂₆—(CH₂)e—, eis 2-4, and R₂₆ is alkoxy, lower alkyl sulfonyl, loweralkylthio, orNHR₂₉ where R₂₉ is loweralkoxycarbonyl or loweralkylaminocarbonyl, andthe dotted bond is hydrogenated.
 57. A compound of claim 56 wherein R₂₆is methoxy, methyl sulfonyl, or methylthio.
 58. A compound of claim 50wherein Y is Y-1

where R₂₂ is hydrogen or lower alkyl, R₂₃ is halogen, lower alkyl, orperfluoroalkyl, and R₂₄ is hydrogen.
 59. A compound of claim 58 whereinR₁₅ is chlorine and R₁₆ is hydrogen or chlorine.
 60. A compound of claim59 which is4-[[(2,6-dichlorophenyl)carbonyl]amino]-N-[(2-bromophenyl)thioxomethyl]-L-phenylalanine.61. A compound of claim 59 which is4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]L-phenylalanine.62. A compound of claim 59 which is4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[(2-fluorophenyl)thioxomethyl]-L-phenylalanine.63. A compound of claim 59 which is4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[2-(trifluoromethyl)phenyl]thioxomethyl]-L-phenylalanine.64. A compound of claim 52 wherein Y is Y-1

where R₂₂ is hydrogen or lower alkyl, R₂₃ is halogen, lower alkyl, orperfluoroalkyl, and R₂₄ is hydrogen.
 65. A compound of claim 53 whereinY is Y-1

where R₂₂ is hydrogen or lower alkyl, R₂₃ is halogen, lower alkyl, orperfluoroalkyl, and R₂₄ is hydrogen.
 66. A compound of claim 65 which is4-[(2S,4R)-3-acetyl-2-phenyl-4-[(3-pyridinyl)methyl]-5-oxo-imidazolidin-1-yl]-N-[(2-ethyl-6-methylphenyl)thioxomethyl]-L-phenylalanine.67. A compound of claim 50 wherein Y is Y-3

which is a four to six membered cycloalkyl ring, R₂₅ is R₂₆—(CH₂)e—, eis 2-4, and R₂₆ is alkoxy, lower alkyl sulfonyl, loweralkylthio, orNHR₂₉ where R₂₉ is loweralkoxycarbonyl or loweralkylaminocarbonyl, andthe dotted bond is hydrogenated.
 68. A compound of claim 67 wherein R₁₅is chlorine and R₁₆ is hydrogen or chlorine.
 69. A compound of claim 68which is4-[[2,6-dichlorophenyl)carbonyl]amino-N-[[1-[2-(acetylamino)ethyl]cyclopentyl]thioxomethyl]-L-phenylalanine.70. A compound of claim 68 which is[[1-[2-[[(methylamino)carbonyl]amino]ethyl]cyclopentyl]thioxomethyl]-4-[[(2,6-dichlorophenyl)carbonyl]amino]L-phenylalanine.71. A compound of claim 68 which is4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1-(2-methoxyethyl)cyclopentyl]thioxomethyl]-L-phenylalanine.72. A compound of claim 68 which is4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1-[(4-methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.73. A compound of claim 68 which is4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1-(3-methylthio)propyl]cyclobutyl]thioxomethyl]-L-phenylalanine.74. A compound of claim 68 which is4-[[(2,6,-dichlorophenyl)carbonyl]amino]-N-[[1-(3-methylsulfonyl)propyl]cyclobutyl]thioxomethyl]-L-phenylalanine.75. A compound of claim 67 wherein R26 is methoxy, methyl sulfonyl, ormethyl thio.
 76. A compound of claim 68 wherein R26 is methoxy, methylsulfonyl, or methyl thio.
 77. A compound of claim 52 wherein Y is Y-3

which is a four to six membered cycloalkyl ring, R₂₅ is R₂₆—(CH₂)e—, eis 2-4, and R₂₆ is alkoxy, lower alkyl sulfonyl, loweralkylthio, orNHR₂₉ where R₂₉ is loweralkoxycarbonyl or loweralkylaminocarbonyl, andthe dotted bond is hydrogenated.
 78. A compound of claim 77 wherein R₂₆is methoxy, methyl sulfonyl, or methyl thio.
 79. A compound of claim 78which is4-[(2,6-dimethyl-3-pyridinylcarbonyl)amino]-N-[[1-[(4-methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine.80. A compound of claim 78 which is4-[[[4-(trifluoromethyl)-5-pyrimidinyl]carbonyl]amino]-N-[[1-(4-methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.81. A compound of claim 78 which is4-[[(2,4-dimethyl-6-trifluoromethyl-3-pyridinyl)carbonyl]amino]-N-[[1-[(4-methylsulfonyl)butyl]cyclobutyl]thioxomethyl]-L-phenylalanine.82. A compound of claim 53 wherein Y is Y-3

which is a four to six membered cycloalkyl ring, R₂₅ is R₂₆—(CH₂)e—, eis 2-4, and R₂₆ is alkoxy, lower alkyl sulfonyl, loweralkylthio, orNHR₂₉ where R₂₉ is loweralkoxycarbonyl or loweralkylaminocarbonyl, andthe dotted bond is optionally hydrogenated.
 83. A compound of claim 82wherein R₂₆ is methoxy, methyl sulfonyl, or methyl thio.
 84. A compoundof claim 83 which is4-[(2S,4R)-3-acetyl-2-phenyl-4-[(3-phenyl)methyl]-5-oxo-imidazolidin-1-yl]-N-[[(4-methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine.85. A compound of claim 83 which is4-[(2R,4R)-3-acetyl-2-phenyl-4-[(3-phenyl)methyl]-5-oxo-imidazolidin-1-yl]-N-[[(4-methylsulfonyl)butyl]cyclopentyl]thioxomethyl]-L-phenylalanine.